Enzymes with xylanase activity from aspergillus aculeatus

ABSTRACT

An enzyme preparation comprising (a) first enzyme with xylanase activity and (b) a second enzyme having cellulotytic, xylanolytic or pectinolytic activity and are supported by the specification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/219,277,filed Dec. 22, 1998, which is a continuation of Ser. No. 09/116,622filed Jul. 16, 1998, now U.S. Pat. No. 6,080,567, which is acontinuation of application Ser. No. 08/902,655, filed Jul. 30, 1997,now U.S. Pat. No. 5,885,819, which is a divisional application of Ser.No. 08/507,431, filed Feb. 15, 1996, now U.S. Pat. No. 5,693,518, whichis a 35 U.S.C. 371 national application of PCT/DK94/00088 and claimspriority under 35 U.S.C. 119 of Danish application Ser. Nos. 0268/93filed on Mar. 10, 1993 and 1151/93 filed on Oct. 14, 1993, the contentsof which are fully incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to an enzyme with xylanase activity, amethod of producing the enzyme, an enzyme preparation containing theenzyme, and use of the enzyme for various industrial purposes.

BACKGROUND OF THE INVENTION

Xylan, a major component of plant hemicellulose, is a polymer ofD-xylose linked by beta-1,4-xylosidic bonds. Xylan can be degraded toxylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatichydrolysis of xylan produces free sugars without the by-products formedwith acid (e.g. furans).

Enzymes which are capable of degrading xylan and other plant cell wallpolysaccharides are important for the food industry, primarily forbaking and in fruit and vegetable processing such as fruit juiceproduction or wine making, where their ability to catalyse thedegradation of the backbone or side chains of the plant cell wallpolysaccharide is utilised (Visser et al., Xylans and.Xylanases, 1991).

Other applications for xylanases are enzymatic breakdown of agriculturalwastes for production of alcohol fuels, enzymatic treatment of animalfeeds for hydrolysis of pentosans, manufacturing of dissolving pulpsyielding cellulose, and bio-bleaching of wood pulp (Detroym R. W. In:Organic Chemicals from Biomass, (CRC Press, Boca Raton, Fla., 1981)19-41.; Paice, M. G., and L. Jurasek., J. Wood Chem. Technol. 4:187-198.; Pommier, J. C., J. L. Fuentes, G. Goma., Tappi Journal (1989):187-191.; Senior, D. J., et al., Biotechnol. Letters 10 (1988):907-912].WO 92/17573 discloses a substantially pure xylanase derived from thefungal species H. insolens and recombinant DNA encoding said xylanase.The xylanase is stated to be useful as a baking agent, a feed additive,and in the preparation of paper and pulp.

WO 92/01793 discloses a xylanase derived from the fungal speciesAspergillus tubigensis. It is mentioned, but not shown that relatedxylanases may be derived from other filamentous fungi, examples of whichare Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarlum andTrichoderma. The xylanases are stated to be useful in the preparation ofbread or animal feed, in breewing and in reducing viscosity or improvingfilterability of cereal starch.

Shei et al., 1985, and Fournier et al., 1985 describe purification andcharacterization of endoxylanases isolated from A. niger.

WO 91/19782 and EP 463 706 discloses xylanase derived from Aspergillusniger origin and the recombinant production thereof. The xylanase isstated to be useful for baking, brewing, in the paper making industry,and in the treatment of agricultural waste, etc.

SUMMARY OF THE INVENTION

It is an object of the present invention to prepare single-componentxylanases.

Accordingly, the present invention relates to an enzyme exhibitingxylanase activity, which enzyme is immunologically reactive with anantibody raised against a purified xylanase derived from Aspergillusaculeatus, CBS 101.43.

In the present context, the term “derived from” is intended not only toindicate a xylanase produced by strain CBS 101.43, but also a xylanaseencoded by a DNA sequence isolated from strain CBS 101.43 and producedin a host organism transformed with said DNA sequence.

In another aspect, the invention relates to an enzyme exhibitingxylanase activity, which enzyme is encoded by a DNA sequence comprisingat least one of the following partial sequences

(a) CATCAACATT CATTCATTCA (SEQ ID No. 7) (b) TTTAATTCAT TCCTCAAGCT (SEQID No. 8) (c) CAAGAGCAGT CATCCCTTCT (SEQ ID No. 9) (d) TTCCAACATGGTTCAAATCA (SEQ ID No. 10) (e) AAGCAGCTGC TCTGGCTGTC (SEQ ID No. 11) (f)CTTTTCGCCA GCAATGTGCT (SEQ ID No. 12) (g) CTCCAACCCC ATCGAGCCCCG (SEQ IDNo. 13) (h) CCAGGCCTCG GTGAGCATCGA (SEQ ID No. 14) (i) TGCCAAATTACAAGGCGCACG (SEQ ID No. 15) (j) CAAGAAGTAC CTGGGCACCAT (SEQ ID No. 16)(k) GAACCCCCAC AATCACGCAA (SEQ ID No. 17) (l) AAATGGTCGG ACTGCTTTCA (SEQID No. 18) (m) ATCACCGCGG CGCTTGCCG (SEQ ID No. 19) (n) CTGTGTTGCCAAACATTGTC (SEQ ID No. 20) (o) TCTGCCGTTG GTCTGGATCA (SEQ ID No. 21) (p)GGCTGCAGTT GCCAAAGGAC (SEQ ID No. 22) (q) TTCAATACTT TGGCACAGCT (SEQ IDNo. 23) (r) ACGGATAATC CCGAGCTCAC (SEQ ID No. 24) (s) GGATATTCCATACGTTACTCA (SEQ ID No. 25) (t) GCTGAACAAC ACCGCGGACT (SEQ ID No. 26) (u)TTGGTCAAAT TACCCCTGGAAAC (SEQ ID No. 27) (v) TCGATGAAGT GGGATGCCAC (SEQID No. 28) (w) AGAACCATCT CAGGGCACCTTC (SEQ ID No. 29) (x) ACGTTCACGAAAGGC (SEQ ID No. 30) (y) CTTCTACTTA GTATTCA (SEQ ID No. 31) (z)CTGACTTACC ATGGCTCGCC (SEQ ID No. 32) (A) TATCTCAGTT CCTTCTGGCC (SEQ IDNo. 33) (B) TGCGCTCTTG CAGTCAAAG (SEQ ID No. 34) (C) CCTTCGCTGCCCCCGCCGCC (SEQ ID No. 35) (D) GAGCCCGTCG AGGAACGGGG (SEQ ID No. 36) (E)CCCTAACTTC TTTTCTGCCC (SEQ ID No. 37) (F) TTGCTGGGCG CTCGACTGG (SEQ IDNo. 38) (G) CAGCTCCACT GGCTACTCGAA (SEQ ID No. 39)

In further aspects the invention relates to an enzyme exhibitingxylanase activity, which enzyme is encoded by a DNA sequence comprisedin or comprising a DNA sequence shown in any of SEQ ID Nos. 1, 3 or 5,respectively, or sequence homologous thereto encoding a polypeptide withxylanase activity.

The enzyme encoded by the DNA sequence shown in SEQ ID No. 1 is termedxylanase I (or xyl I) in the following disclosure, the enzyme encoded bythe DNA sequence SEQ ID No. 3 is termed xylanase II (or xyl II) in thefollowing disclosure, and the enzyme encoded by the DNA sequence SEQ IDNo. 5 is termed xylanase III (or xyl III) in the following disclosure.

In a further aspect, the invention relates to an enzyme exhibitingxylanase activity, which enzyme is encoded by a DNA sequence comprisingthe following partial sequence

(SEQ ID No. 40) CATCAACATT CATTCATTCA TTTAATTCAT TCCTCAAGCT CAAGAGCAGTCATCCCTTCT TTCCAACATG GTTCAAATCA AAGCAGCTGC TCTGGCTGTC CTTTTCGCCAGCAATGTGCT CTCCAACCCC ATCGAGCCCC GCCAGGCCTC GGTGAGCATC GATGCCAAATTCAAGGCGCA CGGCAAGAAG TACCTGGGCA CCAT

or a sequence homologous thereto encoding a polypeptide with xylanaseactivity. A particular example of such enzyme is xylanase I as definedabove.

In a still further aspect, the invention relates to an enzyme exhibitingxylanase activity, which enzyme is encoded by a DNA sequence comprisingthe following partial sequence

(SEQ ID NO. 41) AAAATGGTCG GACTGCTTTC AATCACCGCG GCGCTTGCCG CGACTGTGTTGCCAAACATT GTCTCTGCCG TTGGTCTGGA TCAGGCTGCA GTTGCCAAAG GACTTCAATACTTTGGCACA GCTACGGATA ATCCCGAGCT CACGGATATT CCATACGTTA CTCAGCTGAACAACACCGCG GACTTTGGTC AAATTACCCC TGGAAACTCG ATGAAGTGGG ATGCCACAGAACCATCTCAG GGCACCTTCA CGTTCACGAAAGGCG

or a sequence homologous thereto encoding a polypeptide with xylanaseactivity. A particular example of such enzyme is xylanase II as definedabove.

In a still further aspect, the invention relates to an enzyme exhibitingxylanase activity, which enzyme is encoded by a DNA sequence comprisingthe following partial sequence

(SEQ ID No. 42) TCCCTTCTAC TTAGTATTCA CTGACTTACC ATGGCTCGCC TATCTCAGTTCCTTCTGGCC TGCGCTCTTG CAGTCAAAGC CTTCGCTGCC CCCGCCGCCG AGCCCGTCGAGGAACGGGG CCTAACTTCT TTTCTGCCCT TGCTGGGCGC TCGACTGGCA GCTCCACTGGCTACTCGAA

or a sequence homologous thereto encoding a polypeptide with xylanaseactivity. A particular example of such enzyme is xylanase III as definedabove.

In the present context, the term “homologue” is intended to indicate apolypeptide encoded by DNA which hybridizes to the same probe as; theDNA coding for the xylanase enzyme under certain specified conditions(such as presoaking in SXSSC and prehybridizing for 1 h at ˜40° C. in asolution of 5×SSC, 5×Denhardt's solution, 50 mM sodium phosphate, pH6.8, and 50 μg of denatured sonicated calf thymus DNA, followed byhybridization in the same solution supplemented with 50 μCi 32-P-dCTPlabelled probe for 18 h at ˜40° C. followed by washing three times in2×SSC, 0.2% SDS at 40° C. for 30 minutes). More specifically, the termis intended to refer to a DNA sequence which is at least 70% homologousto any of the sequences shown above encoding a xylanase of theinvention, such as at least 75%, at least 80%, at least 85%, at least90% or even at least 95% homologous to any of the sequences shown above.The term is intended to include modifications of any of the DNAsequences shown above, such as nucleotide substitutions which do notgive rise to another amino acid sequence of the xylanase, but whichcorrespond to the codon usage of the host organism into which the DNAconstruct is introduced or nucleotide substitutions which do give riseto a different amino acid sequence and therefore, possibly, a differentprotein structure which might give rise to a xylanase mutant withdifferent properties than the native enzyme. Other examples of possiblemodifications are insertion of one or more nucleotides into thesequence, addition of one or more nucleotides at either end of thesequence, or deletion of one or more nucleotides at either end or withinthe sequence.

In a still further aspect, the present invention relates to an enzymepreparation useful for the degradation of plant cell wall components,said preparation being enriched in an enzyme exhibiting xylanaseactivity as described above.

In final aspects the invention relates to the use of an enzyme or enzymepreparation of the invention for various industrial applications.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment the enzyme of the invention comprises 30 or iscomprised in the amino acid sequence apparent from SEQ ID Nos. 2, 4 and6, respectively, or an analogous sequence thereof. The amino acidsequences shown in these SEQ ID's have been deduced from the DNAsequences shown in SEQ ID Nos. 1, 3 and 5, respectively, encodingxylanase I, II and III as defined above.

In the present context, the term “analogous sequence” is intended toindicate an amino acid sequence differing from that of SEQ ID No. 2, 4and 6, respectively, by one or more amino acid residues. The analogoussequence may be one resulting from modification of an amino acidsequence shown in these SEQ ID's, e.g. involving substitution of one ormore amino acid residues at one or more different sites in the aminoacid sequence, deletion of one or more amino acid residues at either orboth ends of the enzyme or at one or more sites in the amino acidsequence, or insertion of one or more amino acid residues at one or moresites in the amino acid sequence. The modification of the amino acidsequence may suitably be performed by modifying the DNA sequenceencoding the enzyme, e.g. by site-directed or by random mutagenesis or acombination of these techniques in accordance with well-knownprocedures. Alternatively, the analogous sequence may be one of anenzyme derived from another origin than the xylanase corresponding toSEQ ID Nos. 2, 4 and 6, respectively.. The analogous sequence willnormally exhibit a degree of homology (in terms of identity) of at least70%, such as at least 75%, 80%, 85%, 90% or even 95% with the amino acidsequence shown in SEQ ID Nos. 2, 4 and 6, respectively.

It has surprisingly been found that xylanase II of the present inventionin addition to xylanase activity exhibits α-arabino-pyranosidaseactivity.

The enzyme of the invention may be isolated by a general methodinvolving

cloning, in suitable vectors, a DNA library from Aspergillus aculeatus,

transforming suitable yeast host cells with said vectors,

culturing the host cells under suitable conditions to express any enzymeof interest encoded by a clone in the DNA library, and

screening for positive clones by determining any xylanase activity ofthe enzyme produced by such clones.

A more detailed description of this screening method is given in Example1 below. expressing the appropriate enzyme activity (i.e. xylanaseactivity as defined by the ability of the enzyme to hydrolyse glycosidicbonds in xylan). The appropriate DNA sequence may then be isolated fromthe clone by standard procedures, e.g. as described in Example 1. It isexpected that a DNA sequence coding for a homologous enzyme may bederived by similarly screening a cDNA library of another microorganism,in particular a fungus, such as a strain of another Aspergillus sp., inparticular a strain of A. aculeatus or A. niger, a strain of aTrichoderma sp., in particular a strain of T. harzianum, or T. reesie, astrain of a Fusarium sp., in particular a strain of F. oxysporum, or astrain of a Humicola sp. or a strain of Scytallidium sp.

Alternatively, the DNA coding for an xylanase of the invention may, inaccordance with well-known procedures, conveniently be isolated from DNAfrom any of the above mentioned organisms by use of syntheticoligonucleotide probes prepared on the basis of a DNA or amino acidsequence disclosed herein. For instance, a suitable oligonucleotideprobe may be prepared on the basis of any of the partial nucleotidesequences (a)-(G) listed above.

The DNA sequence may subsequently be inserted into a recombinantexpression vector. This may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the xylanase should be operablyconnected to a suitable promoter and terminator replicated together withthe chromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the xylanase should be operablyconnected to a suitable promoter and terminator sequence. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. The procedures used toligate the DNA sequences coding for the xylanase, the promoter and theterminator, respectively, and to insert them into suitable vectors arewell known to persons. skilled in the art (cf., for instance, Sambrooket al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,N.Y., 1989).

The host cell which is transformed with the DNA sequence encoding theenzyme of the invention is preferably a eukaryotic cell, in particular afungal cell such as a yeast or filamentous fungal cell. In particular,the cell may belong to a species of Aspergillus, most preferablyAspergillus oryzae or Aspergillus niger. Fungal cells may be transformedby a process involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall in a manner knownper se. The use of Aspergillus as a host microorganism is described inEP 238 023 (of Novo Nordisk A/S) , the contents of which are herebyincorporated by reference. The host cell may also be a yeast cell, e.g.a strain of Saccharomnyces, in particular Saccharomyces cerevisiae.

In a still further aspect, the present invention relates to a method ofproducing an enzyme according to the invention, wherein a suitable hostcell transf ormed with a DNA sequence encoding the enzyme is culturedunder conditions permitting the production of the enzyme, and theresulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed xylanase may conveniently be secreted into the culture mediumand may be recovered therefrom by wel-lknown procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

The thus purified xylanase may be employed for immunization of animalsfor the production of antibodies. More specifically, antiserum againstthe xylanase of the invention may be raised by immunizing rabbits (orother rodents) according to the a procedure described by N. Axelsen etal. in: A Manual of Quantitative Immunoelectrophoresis, BlackwellScientific Publications, 1973, Chapter 23, or A. Johnstone and R.Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pp. 27-31). Purified immunoglobulins may beobtained from the antisera, for example by salt precipitation ((NH₄)₂SO₄), followed by dialysis and ion exchange chromatography, e.g. onDEAE-Sephadex. Immunochemical characterization of proteins may be doneeither by Outcherlony double-diffusion analysis (O. Ouchterlony in:Handbook of Experimental Immunology (D. M. Weir, Ed.), BlackwellScientific Publications, 1967, pp. 655-706), by crossedimmunoelectrophoresis (N. Axelsen et al., supra, Chapters 3 and 4), orby rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).

In a still further aspect, the present invention relates to an enzymepreparation useful for the degradation of plant cell wall components,said preparation being enriched in an enzyme exhibiting xylanaseactivity as described above. In this manner a boosting of the cell walldegrading ability of the enzyme preparation can be obtained.

The enzyme preparation having been enriched with an enzyme of theinvention may e.g. be an enzyme preparation comprising multipleenzymatic activities, in particular an enzyme preparation comprisingmultiple plant cell wall degrading enzymes such as Pectinex®, PectinexUltra SP®, CELLULAST™ or CELLUZYME™ (all available from Novo NordiskA/S). In the present context, the term “enriched” is intended toindicate that the xylanase activity of the enzyme preparation has beenincreased, e.g. with an enrichment factor of 1.1, conveniently due toaddition of an enzyme of the invention prepared by the method describedabove.

Alternatively, the enzyme preparation enriched in an enzyme exhibitingxylanase activity may be one which comprises an enzyme of the inventionas the major enzymatic component, e.g. a mono-component enzymepreparation.

The enzyme preparation may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry preparation. Forinstance, the enzyme preparation may be in the form of a granulate or amicrogranulate. The enzyme to be included in the preparation may bestabilized in accordance with methods known in the art.

The enzyme preparation of the invention may, in addition to a xylanaseof the invention, contain one or more other plant cell wall degradingenzymes, for instance those with cellulytic, xylanolytic or pectinolyticactivities such as α-arabinosidase, α-glucoronisidase, β-xylosidase,xylan acetyl esterase, arabinanase, rhamnogalacturonase, pectinacetylesterase, galactanase, polygalacturonase, pectin lyase, pectatelyase, glucanase or pectin methylesterase. The additional enzyme(s) maybe producible by means of a microorganism belonging to the genusAspergillus, preferably Aspergillus niger, Aspergillus aculeatus,Aspergillus awamori or Aspergillus oryzae, or Trichoderma.

The enzyme preparation according to the invention is preferably used asan agent for degradation or modification of plant cell walls or anyxylan-containing material originating from plant cells walls due to thehigh plant cell wall degrading activity of the xylanase of theinvention.

Examples are given below of preferred uses of the enzyme preparation ofthe invention. The dosage of the enzyme preparation of the invention andother conditions under which the preparation is used may be determinedon the basis of methods known in the art.

The xylanases of the invention hydrolysis β-1,4 linkages in xylans.Xylans are polysaccharides having a backbone composed of β-1,4 linkedxylose. The backbone may have different sidebranches, like arabinose,acetyl, glucuronic acid, 4-ethylglucuronic acid sidebranches. Thecomposition and number of sidebranches vary according to the source ofthe xylan. Arabinose sidebranches dominate in xylans from cerealendosperm, whereas xylans from hard wood contain relatively more acetyland glucuronic acid substituents (Michael P. Coughlan and Geoffrey P.Hazlewood. Biotechnol. Appl. Biochem. 17 : 259-289 (1993). Xylanoriginating from red algae contains a mixture of β-1,4 and β-1,3 linkedxylose in the backbone, this type of xylan is degradable by xylanases tovarying extent due to the 1,4-links in the backbone.

The degradation of xylan by xylanases is facilitated by full or partialremoval of the sidebranches. Acetyl groups can be removed by alkali, orby xylan acetyl-esterases, arabinose sidegroups can be removed by a mildacid treatment or by alpha-arabinosidases and the glucuronic acidsidebranches can be removed by alpha-glucuronisidases. The oligomerswith are released by the; xylanases, or by a combination of xylanasesand sidebranch-hydrolysing enzymes as mentioned above can be furtherdegraded to free xylose by beta-xylosidases.

Xylanases of the present invention can be used without other xylanolyticenzymes or with limited activity of other xylanolytic enzymes to degradexylans for production of oligosaccharides. The oligosaccharides may beused as bulking agents, like arabinoxylan oligosaccharides released fromcereal cell wall material, or of more or less purified arabinoxylansfrom cereals. Xylanases of the present invention can be used incombination of other xylanolytic enzymes to degrade xylans to xylose andother monosaccharides. The released xylose may be converted to othercompounds like furanone flavours.

Xylanases of the present invention may be used alone or together withother enzymes like glucanases to improve the extraction of oil fromoil-rich plant material, like corn-oil from corn-embryos.

Xylanases of the present invention may be used in baking so as toimprove the development, elasticity and/or stability of dough and/or thevolume, crumb structure and/or anti-staling properties of the bakedproduct. Although the xylanases may be used for the preparation of doughor baked products prepared from any type of flour or meal (e.g. based onrye, barley, oat, or maize) xylanases of the invention have been foundto be particularly useful in the preparation of dough or baked productsmade from wheat or comprising substantial amounts of wheat. The bakedproducts produced with an xylanase of the invention includes bread,rolls, baquettes and the like. For baking purposes the xylanase of theinvention may be used as the only or major enzymatic activity, or. maybe used in combination with other enzymes such as a lipase, an amylase,an oxidase (e.g. glucose oxidase, peroxidase), a laccase and/or aprotease.

Xylanases of the present invention may be used for modification ofanimal feed and may exert their effect either in vitro (by modifyingcomponents of the feed) or in vivo. The xylanases are particularlysuited for addition to animal feed compositions containing high amountsof arabinoxylans and glucuronoxylans, e.g. feed containing cereals suchas barley, wheat, rye or oats or maize. When added to feed the xylanasesignificantly improves the in vivo break-down of plant cell wallmaterial partly due to a reduction of the intestinal viscosity (Bedfordet al., 1993), whereby a better utilization of the plant nutrients bythe animal is achieved. Thereby, the growth rate and/or feed conversionratio (i.e. the weight of ingested feed relative to weight gain) of theanimal is improved. The use of a xylanase of the invention in thepreparation of feed is illustrated in Example 8.

Xylanases of the present invention may be used in the paper and pulpindustry, inter alia in the bleaching process to enhance the brightnessof bleached pulps whereby the amount of chlorine used in the bleachingstages may be reduced, and to increase the freeness of pulps in therecycled paper process (Eriksson, K. E. L., Wood Science and Technology24 (1990): 79-101; Paice, et al., Biotechnol. and Bioeng. 32 (1988):235-239 and Pommier et al., Tappi Journal (1989): 187-191). Furthermore,the xylanases may be used for treatment of lignocellulosic pulp so as toimprove the bleachability thereof. Thereby the amount of chlorine needto obtain a satisfactory bleaching of the pulp may be reduced. Thetreament of lignocellulosic pulp may, e.g., be performed as described inWo 93/08275, WO 91/02839 and WO 92/03608.

Xylanases of the present invention may be used in beer brewing, inparticular to improve the filterability of wort e.g. containing barleyand/or sorghum malt. The xylanases may be used in the same manner aspentosanases conventionallly used for brewing, e.g. as described byVietor et al., 1993 and EP 227 159. Furthermore, the xylanases may beused for treatment of brewers spent grain,. i.e. residuals from beerwort production containing barley or malted barley or other cereals, soas to improve the utilization of the residuals for, e.g., animal feed.

Xylanases of the present invention may be used for separation ofcomponents of plant cell materials, in particular of cereal componentssuch as wheat components. Of particular interest is the separation ofwheat into gluten and starch, i.e. components of considerable commercialinterest. The separation process may be performed by use of methodsknown in the art, conveniently a so-called batter process (or wetmilling process) performed as a hydroclone or a decanter process. In thebatter process, the starting material is a dilute pumpable dispersion ofthe plant material such as wheat to be subjected to separation. In awheat separation process the dispersion is made normally from wheatflour and water.

Xylanases of the invention may also be used in the preparation of fruitor vegetable juice in order to increase yield, and in the enzymatichydrolysis of various plant cell wall-derived materials or wastematerials, e.g. from paper production, or agricultural residues such aswheat-straw, corn cobs, whole corn plants, nut shells, grass, vegetablehulls, bean hulls, spent grains, sugar beet pulp, and the like.

The plant material may be degraded in order to improve different kindsof processing, facilitate purification or extraction of other componentthan the xylans like purification of beta-glucan or beta-glucanoligomers from cereals, improve the feed value, decrease the waterbinding capacity, improve the degradability in waste water plants,improve the conversion of e.g. grass and corn to ensilage, etc.

Finally, xylanases of the invention may be used in modifying theviscosity of plant cell wall derived material. For instance, thexylanases may be used to reduce the viscosity of feed containing xylan,to promote processing of viscous xylan containing material as in wheatseparation, and to reduce viscosity in the brewing process.

The invention is further described in the accompanying drawing in which

FIG. 1 is a restriction map of plasmid pYHD17,

FIG. 2 a restriction map of plasmid pHD 414,

FIG. 3 the pH optimums for Xyl I, Xyl II and xyl III,

FIG. 4 the temperature optimum for Xyl I, Xyl II and Xyl III,

FIG. 5 the gelf iltration chromatogram for degradation of 1%wheat-arabinoxylan degraded Xyl I,

FIG. 6 the gelfiltration chromatogram for degradation of 5% WIP by XylI,

FIG. 7 the gelfiltration chromatogram for degradation of 1%wheat-arabinoxylan by Xyl II,

FIG. 8 the gelfiltration chromatogram for degradation of 5% WIP by XylII,

FIG. 9 the gelf iltration chromatogram for degradation of 1%wheat-arabinoxylan by Xyl III,

FIG. 10 the gelfiltration chromatogram for degradation of 5% WIP by XylIII.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

EXAMPLES

Materials and Methods

Donor organism: mRNA was isolated from Aspergillus aculeatus, CBS101.43, grown in a soy-containing fermentation medium with agitation toensure sufficient aeration. Mycelia were harvested after 3-5 days'growth, immediately frozen in liquid nitrogen and stored at −80° C.

Yeast strains: The Saccharomyces cerevisiae strain used was yNG231 (MATalpha, leu2, ura3-52, his4-539, pep4-delta 1, cir+) or JG169 (MATα; ura3-52; leu 2-3, 112; his 3-D200; pep 4-113; prc1::HIS3; prb1:: LEU2;cir+).

Construction of an expression plasmid: The commercially availableplasmid pYES II (Invitrogen) was cut with SpeI, filled in with KlenowDNA polymerase+DNTP and cut with Clal. The DNA was size fractionated onan agarose gel, and a fragment of about 2000 bp was purified byelectroelution. The same plasmid was cut with ClaI/PvuII, and a fragmentof about 3400 bp was purified by electroelution. The two fragments wereligated to a blunt-ended SphI/EcoRI fragment containing the yeast TPIpromoter. This fragment was isolated from a plasmid in which the TPIpromoter from S. cerevisiae (cf. T. Albers and G. Kawasaki, J. Mol.AlPl. Genet. 1, 1982, pp. 419-434) was slightly modified: an internalSphI site was removed by deleting the four bp constituting the core ofthis site. Furthermore, redundant sequences upstream of the promoterwere removed by Ball exonuclease treatment followed by addition of aSphI linker. Finally, an EcoRI linker was added at position −10. Afterthese modifications, the promoter is included in a SphI-EcoRI fragment.Its effeciency compared to the original promoter appears to beunaffected by the modifications. The resulting plasmid pYHD17 is shownin FIG. 1.

Preparation of RNase-free glassware, tips and solutions: All glasswareused in RNA isolations was baked at +220° C. for at least 12 h.Eppendorf tubes, pipet tips and plastic columns were treated in 0.1%diethylpyrocarbonate (DEPC) in EtOH for 12 h, and autoclaved. Allbuffers and water (except Tris-containing buffers) were treated with0.1% DEPC for 12 h at 37° C., and autoclaved.

Extraction of total A: The total RNA was prepared by extraction withguanidinium thiocyanate followed by ultracentrifugation through a 5.7MCsCl cushion (Chirgwin et al., 1979) using the following modifications.The frozen mycelia were ground in liquid N₂ to fine powder with a mortarand a pestle, followed by grinding in a precooled coffee mill, andimmediately suspended in 5 vols of RNA extraction buffer (4M GUSCN, 0.5%Na-laurylsarcosine, 25 mM Na-citrate, pH 7.0, 0.1M β-mercaptoethanol).The mixture was stirred for 30 min. at RT° and centrifuged (30 min.,.5000 rpm, RT°, Heraeus Megafuge 1.0 R) to pellet the cell debris. Thesupernatant-was collected, carefully layered onto a 5.7M CsCl cushion(5.7M CsCl, 0.1M EDTA, pH 7.5, 0.1% DEPC; autoclaved prior to use) using26.5 ml supernatant per 12.0 ml CsCl cushion, and centrifuged to obtainthe total RNA (Beckman, SW 28 rotor, 25 000 rpm, RT°, 24h). Aftercentrifugation the supernatant was carefully removed and the bottom ofthe tube containing the RNA pellet was cut off and rinsed with 70% EtOH.The total RNA pellet was transferred into an Eppendorf tube, suspendedin 500 μl TE, pH 7.6 (if difficult, heat occasionally for 5 min at 65°C.), phenol extracted and precipitated with ethanol for 12 h at −200° C.(2.5 vols EtOH, 0.1 vol 3M NaAc, pH 5.2). The RNA was collected bycentrifugation, washed in 70% EtOH, and resuspended in a minimum volumeof DEPC-DIW. The RNA concentration was determined by measuringOD_({fraction (260/280)}).

Isolation of poly(A)⁺RNA: The poly(A)⁺RNAs were isolated byoligo(dT)-cellulose affinity chromatography (Aviv & Leder, 1972).Typically, 0.2 g of oligo(dT) cellulose (Boehringer Mannheim) waspreswollen in 10 ml of 1×column loading buffer (20 mM Tris-Cl, pH 7.6,0.5M NaCl, 1 mM EDTA, 0.1% SDS), loaded onto a DEPC-treated, pluggedplastic column (Poly Prep Chromatography Column, Bio Rad), andequilibrated with 20 ml 1×loading buffer. The total RNA was heated at65° C. for 8 min., quenched on ice for 5 min, and after addition of 1vol 2×column loading buffer to the RNA sample loaded onto the column.The eluate was collected and reloaded 2-3 times by heating the sample asabove and quenching on ice prior to each loading. The oligo(dT) columnwas washed with 10 vols of 1×loading buffer, then with 3 vols of mediumsalt buffer (20 mM Tris-Cl, pH 7.6, 0.1M NaCl, 1 mM EDTA, 0.1% SDS),followed by elution of the poly(A)⁺RNA with 3 vols of elution buffer (10mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to +65° C., bycollecting 500 μl fractions. The OD₂₆₀ was read for each collectedfraction, and the mRNA containing fractions were pooled and ethanolprecipitated at −20° C. for 12 h. The poly(A)⁺RNA was collected bycentrifugation, resuspended in DEPC-DIW and stored in 5-10 μg aliquotsat −80° C.

Northern blot analysis: The poly(A)⁺RNAs (5 μg/sample) from variousmycelia were electrophoresed in 1.2 agarose-2.2 M formaldehyde gels(Sambrook et al., 1989) and blotted to nylon membranes (Hybond-N,Amersham) with 10×SSC (Sambrook et al., 1989) as transfer buffer. Threerandom-primed (Feinberg & Vogelstein, 1983) ³²P-labeled cDNA probes wereused in individual hybridizations: 1) a 1.3 kb Not I-Spe I fragment forpolygalacturonase I from A. aculeatus (described in Danish PatentApplication DK 1545/92), 2) a 1.3 kb Not I-Spe I fragment encodingendoglucanase I from A. aculeatus (described in DK 0419/92) and 3) a 1.2kb Eag I fragment for galactanase I from A. aculeatus (described in WO92/13945). Northern hybridizations were carried out in 5×SSC (Sambrooket al., 1989), 5×Denhardt's solution (Sambrook et al., 1989), 0.5% SDS(w/v) and 100 μg/ml denatured salmon sperm DNA with a probeconcentration of ca. 2 ng/ml for 16 h at 65° C. followed by washes in5×SSC at 65° C. (2×15 min), 2×SSC, 0.5% SDS (1×30 min), 0.2×SSC, 0.5%SDS (1×30 min), and 5×SSC (2×15 min). After autoradiography at −80° C.for 12 h, the probe #1 was removed from the filter according to themanufacturer's instructions and rehybridized with probe #2, andeventually with probe #3. The RNA. ladder from Bethesda ResearchLaboratories was used as a size marker.

CDNA synthesis:

First strand synthesis: Double-stranded cDNA was synthesized from 5 μgof A. aculeatus poly(A)⁺RNA by the RNase H method (Gubler & Hoffman1983, Sambrook et al., 1989) using the hairpin modification. Thepoly(A)⁺RNA (5 μg in 5 μl of DEPC-treated water) was heated at 70° C.for 8 min., quenched on ice, and combined in a final volume of 50 μlwith reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3mM MgCl2, 10 mM DTT, Bethesda Research Laboratories) containing 1 mMeach dNTP (Pharmacia), 40 units of human placental ribonucleaseinhibitor (RNasin, Promega), 10 μg of oligo(dT)₁₂₋₁₈ primer (Pharmacia)and 1000 units of SuperScript II RNase H- reverse transcriptase(Bethesda Research Laboratories). First-strand cDNA was synthesized byincubating the reaction mixture at 45° C. for 1 h.

Second strand synthesis: After synthesis 30 μl of 10 mM Tris-Cl, pH 7.5,1 mM EDTA was added, and the mRNA:cDNA hybrids were ethanol precipitatedfor 12 h at −20° C. by addition of 40 μg glycogen carrier (BoehringerMannheim) 0.2 vols 10M NH₄Ac and 2.5 vols 96% EtOH. The hybrids wererecovered by centrifugation, washed in 70% EtOH, air dried andresuspended in 250 μl of second strand buffer (20 mM Tris-Cl, pH 7.4, 90mM KCl, 4.6 mM MgCl2, 10 mM (NH₄)₂SO₄, 16 μM BNAD⁺) containing 100 μMeach dNTP, 44 units of E. coli DNA polymerase I (Amersham), 6.25 unitsof RNase H (Bethesda Research Laboratories) and 10.5 units of E. coliDNA ligase (New England Biolabs). Second strand cDNA synthesis wasperformed by incubating the reaction tube at 16° C. for 3 h, and thereaction was stopped by addition of EDTA to 20 mM final concentrationfollowed by phenol extraction.

Mung bean nuclease treatment: The double-stranded (ds) cDNA was ethanolprecipitated at −20° C. for 12 h by addition of 2 vols of 96% EtOH, 0.1vol 3M NaAc, pH 5.2, recovered by centrifugation, washed in 70% EtOH,dried (SpeedVac), and resuspended in 30 μl of Mung bean nuclease buffer(30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO4, 0.35 mM DTT, 2% glycerol)containing 36 units of Mung bean nuclease (Bethesda ResearchLaboratories). The single-stranded hair-pin DNA was clipped byincubating the reaction at 30° C. for 30 min, followed by addition of 70μl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanolprecipitation with 2 vols of 96% EtOH and 0.1 vol 3M NaAc, pH 5.2 at−20° C. for 12 h.

Blunt-ending with T4 DNA polymerase: The ds cDNA was bluntended with T4DNA polymerase in 50 μl of T4 DNA polymerase buffer (20 mM Tris-acetate,pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM each dNTP and7.5 units of T4 DNA polymerase (Invitrogen) by incubating the reactionmixture at +37° C. for 15 min. The reaction was stopped by addition ofEDTA to 20 mM final concentration, followed by phenol extraction andethanol precipitation.

Adaptor ligation and size selection: After the fill-in reaction the cDNAwas ligated to non-palindromic BstX I adaptors (1 μg/μl, Invitrogen) in30 μl of ligation buffer (50 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTTL1 mM ATP, 25 μg/ml bovine serum albumin) containing 600 pmol BstX Iadaptors and 5 units of T4 ligase (Invitrogen) by incubating thereaction mix at +16° C. for 12 h. The reaction was stopped by heating at+70° C. for 5 min, and the adapted cDNA was size-fractionated by agarosegel electrophoresis (0.8% HSB-agarose, FMC) to separate unligatedadaptors and small cDNAs. The cDNA was size-selected with a cut-off at0.7 kb, and the cDNA was electroeluted from the agarose gel in 10 mMTris-Cl, pH 7.5, 1 mM EDTA for 1 h at 100 volts, phenol extracted andethanol precipitated at −20° C. for 12 h as above.

Construction of cDNA libraries: The adapted, ds cDNA was recovered bycentrifugation, washed in 70% EtOH and resuspended in 25 ml DIW. Priorto large-scale library ligation, four test ligations were carried out in10 μl of ligation buffer (same as above) each containing 1 μl s cDNA(reaction tubes #1-#3), 2 units of T4 ligase (Invitrogen) and 50 ng(tube #1), 100 ng (tube #2) and 200 ng (tubes #3 and #4) Bst XI cleavedyeast expression vector (either pYES 2.0 vector Invitrogen or yHD13).The ligation reactions were performed by incubation at +16° C. for 12 h,heated at 70° C. for 5 min, and 1 μl of each ligation electroporated(200 Ω, 2.5 kV, 25 μF) to 40 μl competent E. coli 1061 cells (OD600=0.9in 1 liter LB-broth, washed twice in cold DIW, once in 20 ml of 10%glycerol, resuspended in 2 ml 10% glycerol). After addition of 1 ml SOCto each transformation mix, the cells were grown at 37° C. for 1 h , 50μl plated on LB+ampicillin plates (100 μg/ml) and grown at 37° C. for 12h.

Using the optimal conditions a large-scale ligation was set up in 40 μlof ligation buffer containing 9 units of T4 ligase, and the reaction wasincubated at 16° C. for 12 h. The ligation reaction was stopped byheating at 70° C. for 5 min, ethanol precipitated at −20° C. for 12 h,recovered by centrifugation and resuspended in 10 μl DIW. One μlaliquots were transformed into electrocompetent E. coli 1061 cells usingthe same electroporation conditions as above, and the transformed cellswere titered and the library plated on LB+ampicillin plates with5000-7000 c.f.u./plate. To each plate was added 3 ml of medium. Thebacteria were scraped off, 1 ml glycerol was added and stored at −80° C.as pools. The remaining 2 ml were used for DNA isolation. If the amountof DNA was insufficient to give the required number of yeast transformants, large scale DNA was prepared from 500 ml medium (TB) inoculatedwith 50 μl of −80° C. bacterial stock propagated overnight.

Construction of yeast libraries: To ensure that all the bacterial cloneswere tested in yeast, a number of yeast transformants 5 times largerthan the number of bacterial clones in the original pools was set as thelimit.

One μl aliquots of purified plasmid DNA (100 ng/μl) from individualpools were electroporated (200 Ω, 1. 5 kV, 25 μF) into 40 μl competentS. cerevisiae JG 169 cells (OD600=1.5 in 500 ml YPD, washed twice incold DIW, once in cold 1M sorbitol, resuspended in 0.5 ml 1M sorbitol,Becker & Guarante, 1991). After addition of 1 ml 1M cold sorbitol, 80 μlaliquots were plated on SC+glucose −uracil to give 250-400 c.f.u./plateand incubated at 30° C. for 3-5 days.

Construction of an Aspergillus expression vector: the vector pHD414 is aderivative of the plasmid p775 (described in EP 238 023). In contrast tothis plasmid, pHD 414 has a string of unique restriction sites betweenthe promoter and the terminator. The plasmid was constructed by removalof an approximately 200 bp long fragment (containing undesirable REsites) at the 3′end of the terminator, and subsequent removal of anapproximately 250 bp long fragment at the 5′end of the promoer, alsocontaining undesirable sites. The 200 bp region was removed by cleavagewith NarI (positioned in the pUC vector) and XbaI (just 3′ to theterminator), subsequent filling in the enerated ends with Klenow DNApolymerase +DNTP, purification of the vector fragment on gel andreligation of the vector fragment. This plasmid was called pHD413.pHD413 was cut with StuI (positioned in the 5′end of the promoter) andPvuII (in the pUC vector), fractionated on gel and religated. Theplasmid pHD 414 is shown in FIG. 2.

Media

YPD: 10 g yeast extract, 20 g peptone, H₂O to 810 ml. Autoclaved, 90 ml20% glucose (sterile filtered) added.

10×Basal salt: 66.8 g yeast nitrogen base, 100 g succinic acid, 60 gNaOH, H₂O ad 1000 ml, sterile filtered.

SC-URA: 90 ml 10×Basal salt, 22.5 ml 20% casamino acids, 9 ml 1%tryptophan, H₂O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 20%glucose or 20% galactose added.

SC-H broth: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 g/l tryptophan. Autoclaved for 20 min. at 121° C. After autoclaving,10 ml of a 30% galactose solution, 5 ml of a 30% glucose solution and0.4 ml of a 5% threonine solution were added per 100 ml medium.

SC-H agar: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 gll tryptophan, and 20 g/l agar (Bacto). Autoclaved for 20 min. at121° C. After autoclaving, 55 ml of a 22% galactose solution and 1.8 mlof a 5% threonine solution were added per 450 ml agar.

YNB-1 agar: 3.3 g/l KH₂PO₄, 16.7 g/l agar, pH adjusted to 7. Autoclavedfor 20 min. at 121° C. After autoclaving, 25 ml of a 13.6% yeastnitrogen base without amino acids, 25 ml of a 40% glucose solution, 1.5ml of a 1% L-leucine solution and 1.5 ml of a 1% histidine solution wereadded per 450 ml agar.

YNB-1 broth: Composition as YNB-1 agar, but without the agar.

AZCL xylan: birchwood or oat spelt xylan available from Megazyme,Australia.

4-methyl-umbelliferyl-α-arabinopyranoside: avaiable from Sigma.

Transformation of Aspergillus oryzae or Aspergillus niger (generalprocedure)

100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory, 1981) is inoculated with spores of A. oryzae or A.niger and incubated with shaking at 37° C. for about 2 days. Themycelium is harvested by filtration through miracloth and washed with200 ml of 0.6M MgSO₄. The mycelium is suspended in 15 ml of 1.2M MgSO₄.10 mM NaH₂PO₄, pH=5.8. The suspension is cooled on ice and 1 ml ofbuffer containing 120 mg of Novozym® 234, batch 1687 is added. After 5minutes 1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubationwith gentle agitation continued for 1.5-2.5 hours at 37° C. until alarge number of protoplasts is visible in a sample inspected under themicroscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayered with 5 ml of 0.6M sorbitol, 100 mMTris-HCl, pH=7.0. Centrifugation is performed for 15 minutes at 100 gand the protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2M sorbitol, 10 mM Tris-HCl, pH=7.5. 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 minutes at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated.

Finally the protoplasts are resuspended in 0.2-1 ml of STC. 100 μl ofprotoplast suspension is mixed with 5-25 μg of the appropriate DNA in 10μl of STC. Protoplasts are mixed with p3SR2 (an A. nidulans amds genecarrying plasmid). The mixture is left at room temperature for 25minutes. 0.2 ml of 60% PEG 4000 (BDH 29576). 10 mM CaCl₂ and 10 mMTris-HCl, pH=7.5 is added and carefully mixed (twice) and finally 0.85ml of the same solution is added and carefully mixed. The mixture isleft at room temperature for 25 minutes, spun at 2500 g for 15 minutesand the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one moresedimentation the protoplasts are spread on the appropriate plates.Protoplasts are spread on minimal plates (Cove Biochem.Biophys.Acta 113(1966) 51-56) containing 1.0M sucrose, pH=7.0, 10 MM acetamide asnitrogen source and 20 mM CsCl to inhibit background growth. Afterincubation for 4-7 days at 37° C. spores are picked and spread forsingle colonies. This procedure is repeated and spores of a singlecolony after the second reisolation is stored as a defined transformant.

Fed batch fermentation

Fed batch fermentation was performed in a medium comprising maltodextrinas a carbon source, urea as a nitrogen source and yeast extract. The fedbatch fermentation was performed by innoculating a shake flask cultureof A. oryzae host cells in question into a medium comprising 3.5% of thecarbon source and 0.5% of the nitrogen source. After 24 hours ofcultivation at pH 5.0 and 34° C. the continuous supply of additionalcarbon and nitrogen sources were initiated. The carbon source was keptas the limiting factor and it was secured that oxμgen was present inexcess. The fed batch cultivation was continued for 4 days, after whichthe enzymes could be recovered by centrifugation, ultrafiltration, clearfiltration and germ filtration. For application experiments, amylaseactivity was reduced to an insignificant level by purification methodsknown in the art. For characterization, the enzymes were completelypurified by anionexchange chromatographic methods known in the art.

Characterization of an enzyme of the invention

SDS-PAGE Electrophoresis: SDS-PAGE electrophoresis was performed in aMini-Leak 4 electrophoresis unit (Kem-En-Tec, Copenhagen) as a modifiedversion of the Laemli procedure (Laemmli, 1970; Christgau, 1991).Briefly, the separation gel was cast with 12% acrylamide; 0.2% BISacrylamide; 0.1%.SDS; 0.375M Tris pH 8.8; 0.04% APS(ammonium-persulphate) & 0.04% TEMED. After 6-15 hours of polymerizationthe stacking gel was cast with 4.5% w/w Acrylamide; 0.075%BIS-acrylamide; 0.1% SDS; 66.5 mM Tris pH 6.8; 0.4% w/w APS (ammoniumpersulphate) & 0.4% TEMED. The electrode chambers are filled withrunning buffer 25 mM Tris-base; 0.192M glycine & 0.05% SDS, whereafterthe samples containing sample buffer are loaded, and the gel is run at2-4 mA/gel for over-night running and 10-30 mA/gel for fast running. Thegel is subsequently removed and stained by either commassie or silverstaining.

Isoelectric focusing: Isoelectric focusing is carried out on AmpholinePAG plates pH 3.5-9.5 (Pharmacia, Upsala) on a Multiphor electrophoresisunit according to the manufactures instructions. After electrophoresisthe gel is either commassie stained or silver stained.

Commassie and silver staining: The gel is carefully removed from theglass plates and incubated on a slowly rotating shaking table inapproximately 100 ml of the following solutions:

Coomassie staining

1) 30 min in 40% v/v ethanol; 5% v/v-acetic acid

2) 30 min in 40% v/v ethanol; 5% v/v acetic acid+0.1% Commassie R250

3) Destaining in 30 min in 40% v/v ethanol; 5% v/v acetic acid untilbackground is sufficiently reduced.

4) Finally the gel is incubated in preserving.solution : 5% v/v aceticacid; 10% v/v ethanol; 5% v/v glycerol and air dried between two sheetsof cellophane membrane.

Silver staining

1) 30 min in 40% v/v ethanol; 5% v/v acetic acid

2) 20 min in 10% v/v ethanol; 5% v/v acetic acid

3) 20 min in 0.0057% w/v APS (0.25 mM)

4) 60 min in 0.1% w/v AgNO₃

5) For development, the gel is dipped in developer: 0.015% formaldehyde;2% w/v Na₂CO₃ for 30-60 sec. Then the gel is incubated in a second roundof developer until satisfactory staining of the proteins has beenachieved (5-15 min.). Finally the gel is incubated in preservingsolution: 5% v/v acetic acid; 10% v/v ethanol; 5% v/v glycerol and airdried between two sheets of cellophane membrane.

The activities of the enzymes are measured either by the release ofreducing sugars from birch xylan (available from Roth, Karlsruhe,Germany) or by the release of blue colour from AZCL-birch xylan fromMegaZyme.

0.5 ml 0.4% AZCL-substrate suspension is mixed with 0.5 ml 0.1Mcitrate/phosphate buffer of optimal pH and 10 μl of a suitably dilutedenzyme solution is added. Incubations are carried out in EppendorfThermomixers for 15 minutes at 30° C. (if not otherwise specified)before heat-inactivation for 20 minutes at 95° C. Enzyme incubations arecarried out in triplicate. A blank is produced in which enzyme is addedbut inactivated immediately. After centrifugation the absorbance of thesupernatant is measured in microtiter plates at 620 nm and the blank issubtracted.

0.5% solutions of birch xylan (Roth) are made in 0.1M citrate/phosphateof the optimal pH, (if not otherwise specified) 10 μl enzyme suitablydiluted solutions are added to 1 ml of substrate, incubations arecarried out at 30° C. for 15 minutes before heat-inactivation as above.Reducing sugars are determined by reaction, in microtiter plates, with aPHBAH reagent comprising 0.15 g of para hydroxy benzoic acid hydrazide(Sigma H-9882), 0.50 g of potassium-sodium tartrate (Merck 8087) and 2%NaOH solution up to 10.0 ml. Results of blanks are subtracted. Xylose isused as a standard.

pH and temperature optimums are measured on the above mentionedsubstrates. 0.1M citrate/phosphate buffers of varying pH are used fordetermination of pH optimum. 0.1M citrate/phosphate buffers at optimalpH is used for reaction at different temperatures for 15 min. in orderto determine the temperature optimum.

Km and specific activity are measured by carrying out incubations atsubstrate concentrations (S) ranging from 0.025 to 1.5% (birch xylan),measure the reaction rate (v), picture S/v as a function of S, carry outlinear regression analysis, is finding the slope (=1/Vmax) and theintercept (Km/Vmax) and calculating Km and the specific activity(=Vmax/E), where E is the amount of enzyme added.

For gelfiltration chromatography 1% solutions of wheat arabino-xylan(Megazyme) or 5% suspensions of insoluble pentosan from wheat (WIP,produced as described below), respectively, are made in 0.1M acetatebuffer pH 5.5. To 1.5 ml of these substrates 30 μl of the followingenzyme solutions (final concentration) are added: Xylanase I (0.1mg/ml), Xylanase II (0.1 mg/ml) and Xylanase III (0.07 mg/ml).

Incubations are carried out at 30° C. for 0, 10, 30, 60 and 120 minutesbefore heat-inactivation at 95° C. for 20 min. Centrifugation is carriedout and supernatants are analysed by injection into three TSK-columns ina row (PW G4000, PW G3000, PW G2500) and saccharides are eluted with0.4M acetate buffer pH 3.0 at 0.8 ml/min. Eluting saccharides aredetermined by a Shimadzu RI detector and data are collected andprocessed by Dionex software. Dextrans (from Serva) are used asmolecular weight standards. Collection of data is commenced 15 minutesafter injection.

Production of insoluble pentosan (WIP) from wheat flour 150 kg of commonwheat flour was suspended in 450 kg of cold water. The suspension washeated to 60° C. and 600 g of Termamyl 120L® were added. After heatingto 95° C. resulting in gelatinization of the starch fraction, thesuspension was cooled to 60° C. with continued hydrolysis for 180 min.After adjusting the pH to 8.0 using NaOH 300 g of Alcalase 2.4L® wereadded. During hydrolysis of protein under constant stirring, the pH wasmaintained between 7.5 and 8.0 titrating with NaOH. The hydrolysis wascontinued for 120 min. the precipitate was recovered aftercentrifugation, washed with water once and then further washed on a 35μm sieve with cold water to remove all residual soluble material. To theresulting insoluble material up to 20 l of water was added, heated to60° C. and after an adjustment of the pH to 8.0 with NaOH 100 g ofAlcalase 2.4L® were added. The hydrolysis and NaOH-titration werecontinued until no further drop in pH was observed. The material wasthen washed again on a 35 μm sieve until all soluble material wasremoved and, finally, freeze dried.

Determination of FXU (endo-xylanase activity)

The endo-xylanase activity is determined by an assay, in which thexylanase sample is incubated with a remazol-xylan substrate(4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R,Fluka), pH 6.0. The incubation is performed at 50° C. for 30 min. Thebackground of non-degraded dyed substrate is precipitated by ethanol.The remaining blue colour in the supernatant is determinedspectrophotometrically at 585 nm and is proportional to the endoxylanaseactivity. The endoxylanase activity of the sample is determined.relatively to an enzyme standard. The assay is further described in thepublication. AF 293.6/1-GB, available upon request from Novo NordiskA/S, Denmark.

EXAMPLE 1

A library from A. aculeatus consisting of approx. 1.5×10⁶ individualclones in 150 pools was constructed. DNA was isolated from 20 individualclones from the library and subjected to analysis for cDNA insertion.The insertion frequency was found to be >90% and the average insert sizewas approximately 1400 bp.

DNA from some of the pools was transformed into yeast, and 50-100 platescontaining 200-500 yeast colonies were obtained from each pool. After3-5 days of growth, the agar plates were replica plated onto severalsets of agar plates. one set of plates containing 0.1% AZCL xylan(Megazyme, Australia) was then incubated for 3-5 days at 30° C. todetect for xylanase activity. Positive colonies were identified ascolonies surrounded by a blue halo. Alternatively, one set of plates wasthen incubated for 3-5 days at 30° C. before overlayering with a xylanoverlayer gel containing 0.1% AZCL xylan and 1% agarose in a buffer withan appropriate pH. After incubation for 1-2 days at 30° C., positivecolonies were identified as colonies surrounded by a blue halo.Surprisingly, it was found that xylanase II yeast colonies degrades4-methyl-umbelliferyl-α-arabinopyranoside in an overlayer with 0.1Mcitrate buffer, pH 5.0, and 1% agarose resulting in a fluorescent zone.This is the first report of a xylanase having α-arabinopyranosidaseactivity.

Cells from enzyme-positive colonies were spread for single colonyisolation on agar, and an enzyme-producing single colony was selectedfor each of the xylanase-producing colonies identified.

Characterization of positive clones: The positive clones were obtainedas single colonies, the cDNA inserts were amplified directly from theyeast colony using biotinylated polylinker primers, purified by magneticbeads (Dynabead M-280, Dynal) system and characterized individually bysequencing the 5′-end of each cDNA clone using the chain-terminationmethod (Sanger et al., 1977) and the Sequenase system (United StatesBiochemical). The DNA sequences of the enzyme genes are shown in SEQ IDNos. 1, 3 and 5, respectively.

Isolation of a cDNA gene for expression in Aspergillus: In order toavoid PCR errors in the gene to be cloned, the cDNA was isolated fromthe yeast plasmids by standard procedures as described below.

One or more of the xylanase-producing colonies was inoculated into 20 mlYNB-1 broth in a 50 ml glass test tube. The tube was shaken for 2 daysat 30° C. The cells were harvested by centrifugation for 10 min. at 3000rpm.

The cells were resuspended in 1 ml 0.9M sorbitol, 0.1M EDTA, pH 7.5. Thepellet was transferred to an Eppendorf tube, and spun for 30 seconds atfull speed. The cells were resuspended in 0.4 ml 0.9M sorbitol, 0.1MEDTA, 14 mM β-mercaptoethanol. 100 μl 2 mg/ml Zymolase was added, andthe suspension was incubated at 37° C. for 30 minutes and spun for 30seconds. The pellet (spheroplasts) was resuspended in 0.4 ml TE. 90 μlof (1.5 ml 0.5M EDTA pH 8.0, 0.6 ml 2M Tris-Cl pH 8.0, 0.6 ml 10% SDS)was added, and the suspension was incubated at 65° C. for 30 minutes. 80μl 5M KOAc was added, and the suspension was incubated on ice for atleast 60 minutes and spun for 15 minutes at full speed. The supernatantwas transferred to a fresh tube which was filled with EtOH (room temp.)followed by thorough but gentle mixing and spinning for 30 seconds. Thepellet was washed with cold 70% ETOH, spun for 30 seconds and dried atroom temperature. The pellet was resuspended in 50 μl TE and spun for 15minutes. The supernatant was transferred to a fresh tube. 2.5 μl 10mg/ml RNase was added, followed by incubation at 37° C. for 30 minutesand addition of 500 μl isopropanol with gentle mixing. The mixture wasspun for 30 seconds, and the supernatant was removed. The pellet wasrinsed with cold 96% EtOH and dried at room temperature. The DNA wasdissolved in 50 μl water to a final concentration of approximately 100μl/ml.

The DNA was transformed into E. coli. by standard procedures. Two E.coli colonies were isolated from each of the transformations andanalysed with the restriction enzymes HindIII and XbaI which excised theDNA insert. DNA from one of these clones was retransformed into yeaststrain JGl69.

The DNA sequences of several of the positive clones were partiallydetermined. The DNA sequences of three distinct xylanases (xyl I, xyl IIand xyl III) are shown in SEQ ID Nos. 1, 2 and 3, respectively. Thesequences shown in these SEQ ID's comprise a poly-A tail, the positionof possible stop codons are indicated in the respective amino acidsequences shown in SEQ ID Nos. 2, 4 and 6.

EXAMPLE 2

Expression of xylanase

In order to express the genes in Aspergillus, cDNA is isolated from oneor more representatives of each family by digestion with HindIII/XbaI orother appropriate restriction enzymes, size fractionation on a gel andpurification and subsequently ligated to pHD414, resulting in theplasmids pXY-I, pXY-II and pXY-III. After amplification in E. coli, theplasmids are transformed into A. orvzae or A. niger according to thegeneral procedure described in the Materials and Methods section above.

Test of A. oryzae transformants

Each of the transformants were inoculated on FG-4 agar in the centre ofa Petri dish. After 5 days of incubation at 30° C., 4 mm diameter plugswere removed by means of a corkscrew. The plugs were embedded in a xylanoverlayer gel, containing 0.1% AZCL xylan and 1% agarose in a bufferwith an appropriate pH, and incubated overnight at 40° C. The xylanaseactivity was identified as described above. Some of the transformantshad halos which were significantly larger than the Aspergillus oryzaebackground. This demonstrates efficient expression of xylanase inAspergillus oryzae. The 8 transformants with the highest xylanaseactivity were selected and inoculated and maintained on YPG-agar.

Each of the 8 selected transformants were inoculated from YPG-agarslants on 500 ml shake flask with FG-4 and MDU-2 media. After 3-5 daysof fermentation with sufficient agitation to ensure good aeration, theculture broths were centrifuged for 10 minutes at 2000 g and thesupernatants were analyzed.

A volume of 15 μl of each supernatant was applied to 4 mm diameter holespunched out in a 0.1% AZCL xylan overlayer gel (25 ml in a 13 cmdiameter Petri dish). The xylanase activity was identified by theformation of a blue halo on incubation.

Subsequently, Xyl I, Xyl II and Xyl III, respectively, were produced byfed batch fermentation of A. oryzae expressing the enzymes as describedin Materials and Methods above.

EXAMPLE 3

Purification of xylanase I, II, & III

Purification of xylanase I

The culture supernatant from fermentation of Aspergillus oryzaeexpressing the recombinant enzyme is centrifuged and filtered through a0.2 μm filter to remove the mycelia. 35-50 ml of the filteredsupernatant (30-60 mg xylanase I) is ultrafiltrated in a Filtronultracette or Amicon ultrafiltration device with a 10 kDa membrane toachieve 10 fold concentration. This concentrate is diluted 100 times in25 mM Tris pH 8.0 in two successive rounds of ultrafiltration in thesame device. This ultrafiltratred sample is loaded at 1.5 ml/min on aPharmacia HR16/20 Fast Flow Q Sepharose anion exchanger equilibrated in25 EM Tris pH 8.0. After the sample has been applied, the column iswashed with two column volumes 25 mM Tris pH 8.0, and bound proteins areeluted with a linear increasing NaCl gradient from 0 to 0.5M NaCl in 25mM Tris pH 8.0. Xylanase I is not bound to the column and is thuspresent in the wash fraction. The majority of all impurities are boundto the column, and thus Xylanase I from the run-through/wash fraction ismore than 95% pure.

Purification of Xylanase II

The culture supernatant from fermentation of Aspergillus oryzaeexpressing the recombinant enzyme is centrifuged and filtered through a0.2 μm filter to remove the mycelia. 35-50 ml of the filteredsupernatant (30-60 mg xylanase II) is ultrafiltrated in a Filtronultracette or Amicon ultrafiltration device with a 10 kDa membrane toachieve 10 fold concentration. This concentrate is diluted 100 times in20 mM Tris pH 8.0 in two successive rounds of ultrafiltration in thesame device. This ultrafiltratred sample is loaded at 1.5 ml/min on aPharmacia HR16/20 Fast Flow Q Sepharose anion exchanger equilibrated in20 mM Tris pH 8.0. After the sample has been applied, the column iswashed with two column volumes 20 mM Tris pH 8.0, and bound proteins areeluted with a linear increasing NaCi gradient from 0 to 0.6M NaCl in 20mM Tris pH 8.0. Xylanase II elutes in two distinct peaks atapproximately 0.2 & 0.3M NaCl. The enzyme in these two fractions hasslightly different isoelectric points (pI 4.65 and pI 4.5 for the firstand last eluted peak respectively), but no differences in the enzymaticproperties were observed between the two fractions of xylanase

Purification of Xylanase III

The culture supernatant from fermentation of Aspergillus oryzaeexpressing the recombinant enzyme is centrifuged and filtered through a0.2 μm filter to remove the mycelia. 35-50 ml of the filteredsupernatant (30-60 mg xylanase III) is ultrafiltrated in a Filtronultracette or Amicon ultrafiltration device with a 10 kDa membrane toachieve 10 fold concentration. This concentrate is diluted 100 times in25 mM Tris pH 8.0 in two successive rounds of ultrafiltration in thesame device. This ultrafiltratred sample is loaded at 1.5 ml/min on aPharmacia HR16/20 Fast Flow Q Sepharose anion exchanger equilibrated in25 mM Tris pH 8.0. After the sample has been applied, the column iswashed with two column volumes 25 mM Tris pH 8.0, and bound proteins areeluted with a linear increasing NaCl gradient from 0 to 0.6M NaCl in 25mM Tris pH 8.0. Xylanase III in this fraction is not completely pure.Thus, the xylanase III containing fractions were concentrated byultrafiltration in Amicon ultraf iltration device with a 10 kDa membraneto a volume of 4.5 ml and applied to a HR 26/60 Sephacryl S200gelfiltration column in 0.25M ammonium acetate pH 5.5 at a constant flowof 1 ml/min. Xylanase III is eluted as one distinct peak with a purityof more than 95%.

EXAMPLE 4

Characterization of xylanases I, II, III

The xylanases were characterized as described in Materials and Methodsand the main results are apparent from the table below:

Xyl I Xyl II Xyl III MW 32.3 56 24.8 pl 8.82 4.5-4.7 5.7 Km 0.32-0.480.02-0.10 0.01-0.08 Specific activity 147-170 180-204 61-69 Mw wasdetermined by SDS-PAGE.

pH and temperature optimum

The pH optimums of the dif ferent enzymes can be seen in FIG. 3 and 4,respectively. It is seen that all the xylanases have a pH optimum in therange from pH 4-6, xylanase II being the most, acidic and xylanase I themost alkaline. Xylanase II is characterized by having a high temperatureoptimum (70° C.) compared to the other enzymes.

The Km and siecific activity for xylanase I, II and III were determinedas described in the Materials and Methods section above. The standarddeviations on 1/V-max and Km/Vmax obtained from the linear regressionanalysis were used to calculate the intervals for the enzymes apparentfrom the above table.

It is apparent that the xylanases have specific activities in the rangeof 50-250 μmol/min/mg enzyme protein, Xylanase II having the highestactivity.

Gelfiltration analysis

The gelfiltration chromatograms obtained for Xylanase I, II and III,respectively, (using the method disclosed in the Materials and Methodssection above) are shown in FIGS. 5-11.

If the degradation profiles are ordered after decreasing extent ofdegradation of soluble arabinoxylan after 10 minutes of incubation thefollowing order is obtained: Xylanase II, xylanase I and xylanase III.

If on the other hand the amount of solubilized insoluble arabinoxylan(judged from the area of the chromatograms) after 10 minutes isconsidered the order is: Xylanase I, xylanase III, xylanase II.

Furthermore, the enzymes can be divided into enzymes which continuedegradation of xylan and enzymes which stop their degradation after acertain time. On soluble arabinoxylan xylanase II stops, while xylanaseII and III continue the degradation (xylanase I creating large amountsof monomer and dimer and xylanase III being more restricted in itsdegradation pattern).

On soluble arabinoxylan xylanase I does not stop degradation, whilexylanase II and xylanase III do. Xylanase II is special in being veryslow in attacking the insoluble substrate.

From the results it is suggested that the enzymes are divided intodifferent classes. Xyl2 acts very fast on soluble arabino-xylan and veryslow on insoluble arabinoxylan and the degradation stops after a while.Xyl3 is fast in the degradation of insoluble arabinoxylan, but does notdegrade the liberated material extensively. Xyl1 is characterized by anextensive degradation of both soluble and insoluble arabinoxylan tooligomers.

EXAMPLE 5

Viscosity reduction of wheat flour

Different xylanases were tested for their viscosity reducing capabilityin wheat flour termed Fakta Flour (“Luksus hvedemel”, a commercial flourof non-specified type, available from Dagligvaregruppen, DK-7100 Vejle,Denmark). The flour had the following composition:

Component (in pct) Fakta flour Protein 10.4 Ash 0.2 Dry substance 10.5Composition of carbohydrates (in PCT): Glucose 97.7 Arabinose 1.1 Xylose0.9 Galactose 0.3

The xylanases tested were

Spezyme CP available from Genencor, USA

a H. insolens xylanase (produced as described in Example 2 of WO92/17573

Xylanase I (produced as described in Example 2 and 3)

Xylanase II (produced as described in Example 2 and 3)

The viscosity reduction was measured by the following method:

100 g of flour is weighed precisely. To 120 ml deionized water held at35° C. the enzymes mentioned above were added. The enzymes are dosed asfollows:

Spezyme CP: 8.5 FXU (corresponding to 3.4 mg protein)

Xylanase I: 28.3 FXU (corresponding to 0.236 mg enzyme protein and 4.2mg protein)

Xylanase II: 7.5 FXU (corresponding to 0.19 mg enzyme protein and 0.25mg protein)

H. insolens xylanase: 82.2 FXU (corresponding to 2.2 mg enzyme proteinand 22.3 mg protein)

A blank sample is used as control (no enzyme added). The flour and waterare stirred by hand for 30 sec and then mixed for precisely 30 sec on ablender (Warring, Commercial laboratory blender, Struers, AdjustmentsOFF 1-7, rotor in bottom (4 blades)) at 7 (maximum speed). It lasts 30sec to pour the liquid into the measuring tube at the viscometer(Programmable rheometer, model DV-111, Brookfield, Spindel 25, themeasuring tube being termostated at 38° C.). The viscosity at 40 rpm ismeasured every 15th sec for 4 minutes. The specific viscosity expressedas mean viscosity of sample/mean viscosity of blank in percents is usedas a measure of the viscosity reduction. The mean viscosity is a mean ofthe level reached after 60 sec and until the end of measurements.

The lowest relative viscosity was found in using xylanase II.

Other xylanases were found to lower the relative viscosity (xylanase I,Spezyme CP) although to a lesser extent. The H. insolens xylanase wasfound to increase the viscosity at this dosage. As an example the abovementioned dosages resulted in specific viscosity of the “Fakta flour” of69% for xylanase II, 78% for xylanase I, 87% for Spezyme CP and 107% forH. insolens xylanase in viscosity percent of blank.

EXAMPLE 6

Wheat separation

The wheat separation capacity of the enzymes mentioned in Example 5 wereevaluated by a centrifugation test. The test was conducted on the flourmentioned in Example 5.

The flour and water were mixed according to the procedure described inExample 5. After blending 10 ml of the batter was centrifugated(Megafuge 1.0 Heraeus Sepatech) at 4332 g for 5 minutes. The starch wasfound in the bottom layer, followed by gluten, sludge and the effluentlayer at the top. The separation is expressed as an effluent percent.The higher percentage the better separation.

It was confirmed that xylanase II performs best. As an example theeffluent of “Fakta flour” was 14% for a blank sample, 21% for SpezymeCP, 22% for xylanase I and 23% for xylanase II.

EXAMPLE 7

Use of xylanase II in the complexing stage of an ozone based bleachingsequence, used for bleaching of a kraft pulp for papermaking

Prior to bleaching with oxμgen containing oxidative bleaching agentssuch as ozone and hydrogen peroxide, kraft pulp is treated in a separatestage with a complexing agent e.g. EDTA or DTPA. The aim is to securethat the subsequent oxidative bleaching is selective towards degradationof the lignin in the pulp fibers. The lignin should be oxidizedselectively because decomposition of cellulose means loss of fiberstrength.

In the complexing stage the concentration of manganese ions bound toorganic acid groups in the fibers are removed to a level ofapproximately 10 ppm. Higher levels of manganese ions would lead to theformation of undesired free radicals with high reactivity on cellulose,thus reducing the selectivity of the bleaching.

Another metal ion present in the pulp, magnesium, is desired to bepresent in high amounts due to a cellulose protective function.Complexing agents will remove some magnesium but by choosing a pH in therange 5 to 7 the complexing stage will remove less than half of theamount of magnesium initially present.

The temperature should be as high as possible but energy considerationsin practice sets an upper limit of 60° C. The optimum for the A.aculeatus II xylanase in bleaching of kraft pulp has been determined tobe 60° C. and pH 5. This xylanase is therefore particularly well suitedfor use in a complexing stage.

This example illustrates how treatment with A. aculeatus xylanase can besuccessfully applied in a complexing stage, simultaneously with acomplexing agent.

A sample of industrial oxygen-delignified softwood kraft pulp wasanalyzed and found to contain 75 ppm manganese and 750 ppm magnesium.The kappa no. was determined to 14.5 according to TAPPI procedure T236.

The bleaching was completed in 4 stages as described below:

Stage 1: EDTA/A. aculeatus xylanase II (OIEnz)

0.8 kg H₂SO₄ and 2 kg EDTA pr. ton oven dry pulp were mixed into thepulp resulting in a pH of 5.0. A. aculeatus xylanase II produced asdescribed in Examples 2 and 3 was then at a dosage of 15.000 FXU per kgoven dry pulp, and the consistency was adjusted to 10% with deionizedwater. The pulp was incubated 60 minutes at 60° C.

After the treatment the concentration of dissolved lignin was determinedas the absorbency at 280 nm of the waterphase (Dence,L: “Methods inlignin Chemistry”, Springer 1992). From initially 1.5 units theabsorbency had risen to 4.6. After washing the pulp the kappa no. wasdetermined to 13.4. The increase in absorbency and the decrease in kappano. show that lignin has been successfully removed from the pulp fibers.

The treated pulp was analyzed for metal ions, the final concentrationswere 10 ppm manganese and 450 ppm magnesium. This shows that thetreatment with EDTA has lead to the desired result, removing most of themanganese and leaving more than half of the magnesium in the pulp.

A reference pulp was treated likewise but without addition of xylanase.This reference pulp had a kappa no. of 14T3 and a content of k ppmmanganese and 450 ppm magnesium.

Another ref erence pulp was treated with xylanase without addition ofEDTA. From initially 1.4 absorb ency units the absorbency had risen to4.5. After washing the pulp the kappa no. was determined to 13.5. Theseresults are essentially the same as for the pulp treated with both EDTAand xylanase.

These results demonstrate how EDTA and treatment with A. aculeatusxylanase II can be carried out at the same time in the same stagewithout interference.

Stage 2: Ozone (Z)

The EDTA and xylanase treated pulp was adjusted to a pH of 2 andbleached in a low-consistency ozone reactor at 25° C., dosing ozone at aslow rate under vigorous mixing until exactlyp 8 kg/ton had beenconsumed. The pulp was then washed with 60° C. water.

Stage 3: oxvgen and Hydragenperoxide reinforced extraction (E_(op))

After washing the pulp was transferred to a pressurized alkalineextraction stage where 0.5% H₂O₂ and 2% NaOH were added and theconsistency was made up to 10%. The pulp was incubated under 4 atm.oxygen 75 minutes in a pressure stainless steel vessel. The pulp waswashed with 60° C. water.

After the wash, the kappa no. was determined to 1.9 compared to 2.8 forthe reference pulp treated without xylanase. The lowef final kappanumber after xylanase pretreatment demonstrates thl improvedbleachability obtained. The brightness (SCAN C11) were 77% ISO for theenzyme treated pulp and 69% ISO for the reference pulp.

Stage 4: Chlorine Dioxide (D)

To obtain full brightness the pulp was finally bleached with chlorinedioxide. After adjusting the consistency to 10%, a dosage of 14.5 kgactive chlorine (or 5.5 kg chlorine dioxide) per ton pulp was added. Thepulp was incubated 3 hours at 60° C. and then washed with water at 60°C.

The final brightness was determined to 90.6% ISO compared to 86.9% ISOobtained for pulp treated without enzyme. The effect of the xylanase hadthus been a 3.7% ISO increase in final brightness.

To indicate the pulp strength the pulp viscosity of the bleached pulpwas determined according to TAPPI T230. The enzyme treated pulp had aviscosity of 20.5 cP, the control treated without xylanase had aviscosity of 19.8 cP. Thus, the EDTA treatment had worked equally wellor better with xylanase present.

EXAMPLE 8

Use of xylanase in animal feed

Broiler chickens were fed for 6 weeks on an experimental diet with andwithout enzymes. The diet contained 81% wheat in the first 3 weeks ofthe trial and 84.5% wheat the last 3 weeks. They were divided into 3treatments; for the first six weeks each treatment included 12repetitions with 8 broilers in each, the last 3 weeks 6 repetitions with5 chickens in each. The treatments included a control without enzymesand the following enzymatic treatments: 400 FXU/kg feed Biofeed Plus(BF+) (available from Novo Nordisk A/S) and 400 FXU/kg feed xylanase II.Both enzymes were formulated as CT granulate according to the methoddescribed in WO 92/12645. Weight gain and feed consumption wasdetermined and feed conversion ration (FCR) was calculated from 0 to 3and from 3 to 6 weeks. Furthermore, jejunal and ileal viscosity wasdetermined on a supernatant from the gut contents, using a BrookfieldLVTDV-II viscosimeter.

The results are apparent from the following tables.

TABLE 1 Production parameters from 0 to 3 weeks. Weight Feed Feed gain/intake/ conversion chick (g) chick (g) (g/g) Control 364.55 647.04 1.78100 BF+ 400 391.88 643.68 1.64 92 Xyl. 2 400 404.83 650.89 1.61 90

TABLE 2 Production parameters from 3 to 6 weeks. Feed Weight Feedconversion gain/ intake/ (g/g) chick (g) chick (g) % Control 835.511882.44 2.22 100 BF+ 400 932.24 1906.70 2.06 93 Xyl. 2 400 1050.082068.44 1.98 89

TABLE 3 Jejunal viscosity at 3 and 6 weeks. 3 weeks 6 weeks Control16.51 6.31 BF+ 400 11.24 12.96 Xyl. 2 400 6.35 3.50

TABLE 4 Ileal viscosity at 3 and 6 weeks. 3 weeks 6 weeks Control 40.0720.41 BF+ 400 18.46 16.92 Xyl. 2 400 15.65 6.27

As can be seen from Tables 1 and 2, the FCR is lower in the groupsreceiving enzymes, both after 3 and 6 weeks. In both cases xylanase IIis better than BF+. This is mainly due to a better growth of the animalsin this group.

With regard to jejunal viscosity xylanase II gives a lower viscositycompared to both BF+ and control. This is also the case for ilealviscosity. Both the control and xylanase II gives a lower viscosityafter 6 weeks than 3 weeks, while this is not the case for BF+. It thusseems-that xylanase II works better during the last 3 weeks than BF+,which is also indicated by the relatively lower FCR of Xylanase IIcompared to BF+ at 6 weeks.

This experiment thus shows that xylanase II gives a better feedconversion than BF+ on the same FXU basis, i.e. that more nutrients aremade available with xylanase II. This may partly be due to a lower ilealviscosity in the xylanase II group.

EXAMPLE 9

Materials and methods

Enzymes

Lipase A: The Humicola lanuginosa lipase described in EP 305 216 andproduced by recombinant DNA techniques in Aspergillus oryzae asdescribed in EP 305 216. The lipase has a specific activity of 4,452,000LU/g and a FAU/g of less than 0.6.

Xylanase A: A xylanase produced by the Humicola insolens strain DSM 1800available from the Deutsche Sammlung von Mikro-organismen undZellkulturen GmbH and further described in EP 507 723.

Fungamyl: A commercial fungal α-amylase preparation available from NovoNordisk A/S, Denmark.

Pentopan: A commercial xylanase preparation available from Novo NordiskA/S, Denmark.

LU/g (Lipase Units/g), FAU/g (Fungal alpha-Amylase Units/g) and FXU(xylanase units/g) were determined by the following assays:

LU—Lipase Units

Lipase activity was assayed using glycerine tributyrat as a substrateand gum-arabic as an emulsifier. 1 LU (Lipase Unit) is the amount ofenzyme which liberates 1 μmol titratable butyric acid per minute at 30°C., pH 7.0. The lipase activity was assayed by pH-stat using Radiometertitrator VIT90, Radiometer, Copenhagen. Further details of the assay aregiven in Novo Analytical Method AF 95/5, available on request.

FAU—Fungal alpha-Amylase Units

1 FA-unit (FAU) is the amount of enzyme which at 37° C. and pH 4.7breaks down 5260 mg of solid starch per hour. Further details of theassay are given in Novo Analytical Method AF 9.⅓, available on request.

FXU—xylanase activity

Was determined as described above.

Preparation of bread

White bread were prepared from the following basic recipe:

Basic recipe Flour (Manitoba) 100% Salt 1.5% Yeast (fresh) 5.0% sugar1.5% Water 58%

The wheat flour was of the type termed “Manitoba” supplied by“Valsemφllerne”, Denmark, October 1993.

Procedure

1. Dough mixing (Spiral mixer)

2 min. at 700 RPM

7 min. at 1400 RPM

the mixing time was determined and adjusted by a skilled baker so as toobtain an optimum dough consistence under the testing conditions used.

2. 1st proof: 30° C.- 80% RH, 16 min.

3. Scaling and shaping;

4. Final proof: 32° C.- 80% RH, 35 min.;

5. Baking: 225° C., 20 min. for rolls and 30 min for loaf.

Evaluation of dough and baked products

Properties of the dough and baked products were determined as follows:

Roll specific volume: the volume of 20 rolls are measured using thetraditional rape seed method. The specific volume is calculated asvolume ml per g bread. The specific volume of the control (withoutenzyme) is defined as 100. The relative specific volume index iscalculated as:$\text{Specific vol. index} = {\frac{\text{specific volume of 20 rolls}}{\text{specific volume of 20 control rolls}} - {*100}}$

Loaf specific volume: the mean value of 4 loaves volume are measuredusing the same methods as described above.

The dough stickiness and crumb structure are evaluated visuallyaccording to the following scale:

Dough stickiness: almost liquid 1 too sticky 2 slightly sticky 3 nicesoft 3.5 normal 4 dry 5 Crumb structure: very poor 1 poor 2 non-uniform3 uniform/good 4 very good 5

The softness of bread crumb is measured by a SMS-Texture Analyzer. Aplunger with a diameter of 20 mm is pressed on the middle of a 20 mmthick slice of bread, The force needed for the plunger to depress thecrumb 5 mm with a speed of 2.0 mm/s is recorded and it is expressed asthe crumb firmness. The lower the value, the softer is the crumb. Fourslices of each bread are measured and the mean value is used.

Xylanase I

The enzyme used was xylanase I, a recombinant A. aculeatus xylanaseproduced in A. oryzae as described in Examples 2 and 3 above. The effectof this xylanase was compared to xylanase A from H. insolens and acommercial available pentosanase, Pentopan. The enzymes were addedeither directly into the baking ingredients mix or it was dispersed inwater before being added to the mix. All tests were carried out in atleast duplicate and the average results were used. The results obtainedare shown in Table 5.

It is apparent from Table 5 that the addition of xylanase I increasesthe volume of rolls orland loaves significantly and the effect is largerthan that obtained by the prior art xylanase A and Pentopan. At theoptimum dosage, i.e. the dosage that gives the maximum specific volumeincrease without getting a too sticky dough, of the known pentosanase(Pentopan) and xylanase (xylanase A) the max. volume increase is about10-16%. At the optimum dosage of Xylanase I (about 350-750 FXU per kgflour) a volume increasing of 29-41% can be achieved without causing atoo sticky dough. With a longer proofing time at 80% RH and 32° C. aneven higher volume increase can be achieved. Furthermore, the crumbstructure and crumb softness upon storage are also improved.

TABLE 5 FXU/kg flour 25 50 100 122 200 350 427 500 750 1000 0 Xylanase IDough stickiness 4 4 3.5 3.5 3.5 3.5 3 2.5 4 Xylanase I SP Volumeindex/- 103 109 111 119 121 129 141 133 100 Rolls Xylanase I SP Volumeindex/-/ 101 102 105 110 110 114 122 121 100 Loaves* Xylanase I SPVolume index/- 134 Loaves** Crumb structure 3 3.5 3.5 3.5 4 3 Crumbfirmness 0.278 0.282 0.206 0.258 0.169 0.173 0.189 0.399 Day 4 0.4340.440 0.345 0.346 0.357 0.320 0.348 0.509 Day 7 0.589 0.551 0.451 0.5610.376 0.383 0.380 0.658 Xylanase A Dough stickiness 3.5 3 SP Volumeindex/- 108 116 Rolls Pentopan Dough stickiness 3.5 3 SP Volume index/-106 111 Rolls *low fermentation time: 35 min. **longer fermentationtime: 50 min.

EXAMPLE 10

In the same manner as described in Example 9, baking trials withxylanase II, a recombinant A. aculeatus xylanase produced as describedin Annex 1, xylanase A and Pentopan were performed. The results obtainedare shown from Table 6.

TABLE 5 FXU/kg flour 25 50 100 122 200 350 427 500 0 Dough stickiness 44 3.5 3-3.5 2.5-3 2.5 Xylanase II SP Volume index/ 111 113 123 124 126134 Rolls SP Volume 103 107 109 113 117 117 index/loaves Crumb structure3 3.5 4 4 4.5 4.5 3 Crumb firmness 0.227 0.193 0.209 0.176 0.215 0.399day 1 Day 4 0.362 0.359 0.358 0.289 0.294 0.509 Day 7 0.435 0.390 0.4330.398 0.386 0.658 Xylanase A Dough stickiness 3.5 3 S.P Volume index/108 116 Rolls Pentopan Dough stickiness 3.5 3 SP Volume index/ 106 111Rolls *low fermentation time: 35 min.

It is apparent from Table 6 that the use of xylanase II increases thevolume of rolls or/and loaves significantly and the effect is largerthan the prior art xylanase and pentosanase. At the optimum dosage ofXylanase II (i.e. about 200 FXU per kg flour) a volume increasing of 24%is achieved without causing a too sticky dough. Furthermore, the crumbstructure and crumb softness upon storage are also improved.

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Visser et al., in “Xylans and Xylanases”, Elsevier Science Publishers,1991.

Vietor et al., 1993, J. Inst. Brew., May-June, 99, pp. 243-248.

42 1273 base pairs nucleic acid single linear DNA (genomic) unknown CDSjoin(68..1048, 1052..1084, 1088..1093, 1097..1102, 1106..1273) 1CATCAACATT CATTCATTCA TTTAATTCAT TCCTCAAGCT CAAGAGCAGT CATCCCTTCT 60TTCCAAC ATG GTT CAA ATC AAA GCA GCT GCT CTG GCT GTC CTT TTC GCC 109 MetVal Gln Ile Lys Ala Ala Ala Leu Ala Val Leu Phe Ala 1 5 10 AGC AAT GTGCTC TCC AAC CCC ATC GAG CCC CGC CAG GCC TCG GTG AGC 157 Ser Asn Val LeuSer Asn Pro Ile Glu Pro Arg Gln Ala Ser Val Ser 15 20 25 30 ATC GAT GCCAAA TTC AAG GCG CAC GGC AAG AAG TAC CTG GGC ACC ATC 205 Ile Asp Ala LysPhe Lys Ala His Gly Lys Lys Tyr Leu Gly Thr Ile 35 40 45 GGC GAC CAG TACACT CTC AAC AAG AAC GCA AAG ACC CCG GCG ATC ATC 253 Gly Asp Gln Tyr ThrLeu Asn Lys Asn Ala Lys Thr Pro Ala Ile Ile 50 55 60 AAG GCC GAC TTT GGCCAG CTG ACT CCG GAG AAC AGC ATG AAG TGG GAT 301 Lys Ala Asp Phe Gly GlnLeu Thr Pro Glu Asn Ser Met Lys Trp Asp 65 70 75 GCT ACT GAG CCC AAC CGAGGA CAG TTC TCC TTC TCG GGG TCG GAT TAC 349 Ala Thr Glu Pro Asn Arg GlyGln Phe Ser Phe Ser Gly Ser Asp Tyr 80 85 90 CTG GTC AAC TTC GCC CAG TCTAAC GGA AAG CTG ATC CGT GGC CAC ACT 397 Leu Val Asn Phe Ala Gln Ser AsnGly Lys Leu Ile Arg Gly His Thr 95 100 105 110 CTC GTC TGG CAC TCA CAGCTC CCG TCC TGG GTG CAG TCC ATC TCC GAT 445 Leu Val Trp His Ser Gln LeuPro Ser Trp Val Gln Ser Ile Ser Asp 115 120 125 AAG AAC ACC CTG ATC CAAGTC ATG CAG AAT CAC ATC ACC ACC GTG ATG 493 Lys Asn Thr Leu Ile Gln ValMet Gln Asn His Ile Thr Thr Val Met 130 135 140 CAG CGC TAC AAG GGC AAGGTC TAC GCC TGG GAC GTT GTC AAT GAG ATC 541 Gln Arg Tyr Lys Gly Lys ValTyr Ala Trp Asp Val Val Asn Glu Ile 145 150 155 TTC AAC GAG GAT GGC TCTCTT TGC CAG AGC CAC TTC TAC AAC GTC ATC 589 Phe Asn Glu Asp Gly Ser LeuCys Gln Ser His Phe Tyr Asn Val Ile 160 165 170 GGT GAG GAC TAT GTG CGCATC GCT TTC GAG ACC GCT CGC GCG GTG GAT 637 Gly Glu Asp Tyr Val Arg IleAla Phe Glu Thr Ala Arg Ala Val Asp 175 180 185 190 CCC AAC GCC AAG CTTTAC ATA AAC GAC TAC AAC CTG GAT TCC GCC TCG 685 Pro Asn Ala Lys Leu TyrIle Asn Asp Tyr Asn Leu Asp Ser Ala Ser 195 200 205 TAC CCG AAA CTG ACCGGC CTG GTC AAC CAC GTC AAG AAG TGG GTC GCA 733 Tyr Pro Lys Leu Thr GlyLeu Val Asn His Val Lys Lys Trp Val Ala 210 215 220 GCT GGC GTC CCC ATCGAC GGA ATT GGT TCC CAA ACC CAC CTG AGC GCG 781 Ala Gly Val Pro Ile AspGly Ile Gly Ser Gln Thr His Leu Ser Ala 225 230 235 GGT GCC GGT GCT GCCGTC TCA GGA GCT CTC AAC GCT CTC GCT GGT GCA 829 Gly Ala Gly Ala Ala ValSer Gly Ala Leu Asn Ala Leu Ala Gly Ala 240 245 250 GGC ACC AAG GAG GTCGCT ATT ACC GAG CTC GAC ATC GCT GGC GCC AGC 877 Gly Thr Lys Glu Val AlaIle Thr Glu Leu Asp Ile Ala Gly Ala Ser 255 260 265 270 TCC ACC GAC TACGTG AAC GTC GTC AAG GCG TGT CTG AAC CAG CCC AAG 925 Ser Thr Asp Tyr ValAsn Val Val Lys Ala Cys Leu Asn Gln Pro Lys 275 280 285 TGC GTC GGT ATCACC GTC TGG GGA AGT TCT GAC CCC GAC TCG TGG CGC 973 Cys Val Gly Ile ThrVal Trp Gly Ser Ser Asp Pro Asp Ser Trp Arg 290 295 300 TCC AGC TCC AGCCCT CTG CTC TTC GAC AGC AAC TAC AAC CCC AAG GCT 1021 Ser Ser Ser Ser ProLeu Leu Phe Asp Ser Asn Tyr Asn Pro Lys Ala 305 310 315 GCT TAT ACC GCTATT GCG AAC GCT CTC TAG TGG TCG TCT CTA TCA CTG 1069 Ala Tyr Thr Ala IleAla Asn Ala Leu Trp Ser Ser Leu Ser Leu 320 325 330 GTA AAG CTC GCA GCTTAA TCT CGG TGA ATC CAG TGA CTG GAA TGT CGT 1117 Val Lys Leu Ala Ala SerArg Ile Gln Leu Glu Cys Arg 335 340 345 CGT GAT CGT AGG ATG AAT ACT CGGGGC TTG CGG GTT GCT TTT TCT GTA 1165 Arg Asp Arg Arg Met Asn Thr Arg GlyLeu Arg Val Ala Phe Ser Val 350 355 360 TTT TCA CCT GAA GTC ATC ATT ATGTTG CTG AAC CTT CCT CTT CTC TTA 1213 Phe Ser Pro Glu Val Ile Ile Met LeuLeu Asn Leu Pro Leu Leu Leu 365 370 375 TTG ATC AAT GGT GAG CAT CGT TTTATT TAT AAA AAA AAA AAA AAA AAA 1261 Leu Ile Asn Gly Glu His Arg Phe IleTyr Lys Lys Lys Lys Lys Lys 380 385 390 AAA AAA AAA AAA 1273 Lys Lys LysLys 395 398 amino acids amino acid linear protein unknown 2 Met Val GlnIle Lys Ala Ala Ala Leu Ala Val Leu Phe Ala Ser Asn 1 5 10 15 Val LeuSer Asn Pro Ile Glu Pro Arg Gln Ala Ser Val Ser Ile Asp 20 25 30 Ala LysPhe Lys Ala His Gly Lys Lys Tyr Leu Gly Thr Ile Gly Asp 35 40 45 Gln TyrThr Leu Asn Lys Asn Ala Lys Thr Pro Ala Ile Ile Lys Ala 50 55 60 Asp PheGly Gln Leu Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr 65 70 75 80 GluPro Asn Arg Gly Gln Phe Ser Phe Ser Gly Ser Asp Tyr Leu Val 85 90 95 AsnPhe Ala Gln Ser Asn Gly Lys Leu Ile Arg Gly His Thr Leu Val 100 105 110Trp His Ser Gln Leu Pro Ser Trp Val Gln Ser Ile Ser Asp Lys Asn 115 120125 Thr Leu Ile Gln Val Met Gln Asn His Ile Thr Thr Val Met Gln Arg 130135 140 Tyr Lys Gly Lys Val Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asn145 150 155 160 Glu Asp Gly Ser Leu Cys Gln Ser His Phe Tyr Asn Val IleGly Glu 165 170 175 Asp Tyr Val Arg Ile Ala Phe Glu Thr Ala Arg Ala ValAsp Pro Asn 180 185 190 Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Leu Asp SerAla Ser Tyr Pro 195 200 205 Lys Leu Thr Gly Leu Val Asn His Val Lys LysTrp Val Ala Ala Gly 210 215 220 Val Pro Ile Asp Gly Ile Gly Ser Gln ThrHis Leu Ser Ala Gly Ala 225 230 235 240 Gly Ala Ala Val Ser Gly Ala LeuAsn Ala Leu Ala Gly Ala Gly Thr 245 250 255 Lys Glu Val Ala Ile Thr GluLeu Asp Ile Ala Gly Ala Ser Ser Thr 260 265 270 Asp Tyr Val Asn Val ValLys Ala Cys Leu Asn Gln Pro Lys Cys Val 275 280 285 Gly Ile Thr Val TrpGly Ser Ser Asp Pro Asp Ser Trp Arg Ser Ser 290 295 300 Ser Ser Pro LeuLeu Phe Asp Ser Asn Tyr Asn Pro Lys Ala Ala Tyr 305 310 315 320 Thr AlaIle Ala Asn Ala Leu Trp Ser Ser Leu Ser Leu Val Lys Leu 325 330 335 AlaAla Ser Arg Ile Gln Leu Glu Cys Arg Arg Asp Arg Arg Met Asn 340 345 350Thr Arg Gly Leu Arg Val Ala Phe Ser Val Phe Ser Pro Glu Val Ile 355 360365 Ile Met Leu Leu Asn Leu Pro Leu Leu Leu Leu Ile Asn Gly Glu His 370375 380 Arg Phe Ile Tyr Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 385 390395 1327 base pairs nucleic acid single linear DNA (genomic) unknown CDSjoin(4..1221, 1225..1314, 1318..1326) 3 AAA ATG GTC GGA CTG CTT TCA ATCACC GCG GCG CTT GCC GCG ACT GTG 48 Met Val Gly Leu Leu Ser Ile Thr AlaAla Leu Ala Ala Thr Val 1 5 10 15 TTG CCA AAC ATT GTC TCT GCC GTT GGTCTG GAT CAG GCT GCA GTT GCC 96 Leu Pro Asn Ile Val Ser Ala Val Gly LeuAsp Gln Ala Ala Val Ala 20 25 30 AAA GGA CTT CAA TAC TTT GGC ACA GCT ACGGAT AAT CCC GAG CTC ACG 144 Lys Gly Leu Gln Tyr Phe Gly Thr Ala Thr AspAsn Pro Glu Leu Thr 35 40 45 GAT ATT CCA TAC GTT ACT CAG CTG AAC AAC ACCGCG GAC TTT GGT CAA 192 Asp Ile Pro Tyr Val Thr Gln Leu Asn Asn Thr AlaAsp Phe Gly Gln 50 55 60 ATT ACC CCT GGA AAC TCG ATG AAG TGG GAT GCC ACAGAA CCA TCT CAG 240 Ile Thr Pro Gly Asn Ser Met Lys Trp Asp Ala Thr GluPro Ser Gln 65 70 75 GGC ACC TTC ACG TTC ACG AAA GGC GAT GTC ATT GCA GATCTG GCT GAG 288 Gly Thr Phe Thr Phe Thr Lys Gly Asp Val Ile Ala Asp LeuAla Glu 80 85 90 95 GGT AAT GGC CAA TAT CTC CGA TGT CAT ACT CTG GTT TGGTAT AAT CAG 336 Gly Asn Gly Gln Tyr Leu Arg Cys His Thr Leu Val Trp TyrAsn Gln 100 105 110 CTA CCT AGC TGG GTG ACT AGC GGA ACT TGG ACT AAT GCTACT CTC ACC 384 Leu Pro Ser Trp Val Thr Ser Gly Thr Trp Thr Asn Ala ThrLeu Thr 115 120 125 GCC GCA TTG AAG AAC CAC ATC ACG AAT GTG GTG TCG CACTAC AAA GGG 432 Ala Ala Leu Lys Asn His Ile Thr Asn Val Val Ser His TyrLys Gly 130 135 140 AAA TGT CTT CAT TGG GAC GTG GTC AAT GAG GCG TTG AATGAC GAC GGA 480 Lys Cys Leu His Trp Asp Val Val Asn Glu Ala Leu Asn AspAsp Gly 145 150 155 ACC TAC CGC ACC AAC ATC TTC TAC ACC ACC ATC GGC GAAGCC TAC ATC 528 Thr Tyr Arg Thr Asn Ile Phe Tyr Thr Thr Ile Gly Glu AlaTyr Ile 160 165 170 175 CCC ATT GCC TTT GCC GCA GCG GCT GCA GCC GAC CCGGAC GCG AAG CTG 576 Pro Ile Ala Phe Ala Ala Ala Ala Ala Ala Asp Pro AspAla Lys Leu 180 185 190 TTC TAC AAT GAC TAC AAC CTC GAA TAC GGC GGC GCCAAA GCC GCC AGC 624 Phe Tyr Asn Asp Tyr Asn Leu Glu Tyr Gly Gly Ala LysAla Ala Ser 195 200 205 GCC CGC GCC ATT GTC CAG CTG GTC AAG AAT GCA GGTGCC AAG ATC GAC 672 Ala Arg Ala Ile Val Gln Leu Val Lys Asn Ala Gly AlaLys Ile Asp 210 215 220 GGG GTA GGG TTG CAG GCC CAT TTC AGC GTC GGC ACCGTG CCG AGT ACG 720 Gly Val Gly Leu Gln Ala His Phe Ser Val Gly Thr ValPro Ser Thr 225 230 235 AGC TCG CTC GTC TCG GTG CTG CAA TCT TTC ACT GCGCTC GGG GTC GAG 768 Ser Ser Leu Val Ser Val Leu Gln Ser Phe Thr Ala LeuGly Val Glu 240 245 250 255 GTC GCC TAC ACG GAG GCC GAC GTG CGC ATT CTCCTG CCC ACC ACC GCC 816 Val Ala Tyr Thr Glu Ala Asp Val Arg Ile Leu LeuPro Thr Thr Ala 260 265 270 ACT ACC CTG GCC CAA CAG TCG AGC GAT TTC CAGGCC CTG GTG CAA TCC 864 Thr Thr Leu Ala Gln Gln Ser Ser Asp Phe Gln AlaLeu Val Gln Ser 275 280 285 TGT GTG CAG ACA ACG GGC TGC GTC GGC TTC ACTATC TGG GAT TGG ACA 912 Cys Val Gln Thr Thr Gly Cys Val Gly Phe Thr IleTrp Asp Trp Thr 290 295 300 GAT AAG TAC AGC TGG GTT CCC AGC ACG TTC TCGGGC TAT GGG GCG GCG 960 Asp Lys Tyr Ser Trp Val Pro Ser Thr Phe Ser GlyTyr Gly Ala Ala 305 310 315 CTA CCC TGG GAT GAG AAC CTG GTT AAG AAG CCCGCG TAC AAT GGC TTG 1008 Leu Pro Trp Asp Glu Asn Leu Val Lys Lys Pro AlaTyr Asn Gly Leu 320 325 330 335 TTG GCC GGC ATG GGG GTT ACA GTT ACC ACTACG ACT ACC ACC ACC ACT 1056 Leu Ala Gly Met Gly Val Thr Val Thr Thr ThrThr Thr Thr Thr Thr 340 345 350 GCT ACT GCC ACT GGT AAG ACT ACG ACT ACCACA ACG GGT GCC ACG AGC 1104 Ala Thr Ala Thr Gly Lys Thr Thr Thr Thr ThrThr Gly Ala Thr Ser 355 360 365 ACG GGG ACT ACG GCT GCG CAT TGG GGG CAGTGT GGA GGG CTC AAC TGG 1152 Thr Gly Thr Thr Ala Ala His Trp Gly Gln CysGly Gly Leu Asn Trp 370 375 380 AGT GGA CCG ACG GCG TGT GCC ACT GGG TACACC TGC ACT TAT GTC AAT 1200 Ser Gly Pro Thr Ala Cys Ala Thr Gly Tyr ThrCys Thr Tyr Val Asn 385 390 395 GAC TAT TAC TCG CAG TGT CTG TGA AGT ATAGCC CAA CCT AAA CCT GCC 1248 Asp Tyr Tyr Ser Gln Cys Leu Ser Ile Ala GlnPro Lys Pro Ala 400 405 410 GGC GTG CTT GCC ATT CAG TCA GTG AGA TTT ATATAT CAC AAT ACT CAA 1296 Gly Val Leu Ala Ile Gln Ser Val Arg Phe Ile TyrHis Asn Thr Gln 415 420 425 430 AAT TCA TTG CTC GAC CTC TGA AAA AAA AAAA 1327 Asn Ser Leu Leu Asp Leu Lys Lys Lys 435 439 amino acids aminoacid linear protein unknown 4 Met Val Gly Leu Leu Ser Ile Thr Ala AlaLeu Ala Ala Thr Val Leu 1 5 10 15 Pro Asn Ile Val Ser Ala Val Gly LeuAsp Gln Ala Ala Val Ala Lys 20 25 30 Gly Leu Gln Tyr Phe Gly Thr Ala ThrAsp Asn Pro Glu Leu Thr Asp 35 40 45 Ile Pro Tyr Val Thr Gln Leu Asn AsnThr Ala Asp Phe Gly Gln Ile 50 55 60 Thr Pro Gly Asn Ser Met Lys Trp AspAla Thr Glu Pro Ser Gln Gly 65 70 75 80 Thr Phe Thr Phe Thr Lys Gly AspVal Ile Ala Asp Leu Ala Glu Gly 85 90 95 Asn Gly Gln Tyr Leu Arg Cys HisThr Leu Val Trp Tyr Asn Gln Leu 100 105 110 Pro Ser Trp Val Thr Ser GlyThr Trp Thr Asn Ala Thr Leu Thr Ala 115 120 125 Ala Leu Lys Asn His IleThr Asn Val Val Ser His Tyr Lys Gly Lys 130 135 140 Cys Leu His Trp AspVal Val Asn Glu Ala Leu Asn Asp Asp Gly Thr 145 150 155 160 Tyr Arg ThrAsn Ile Phe Tyr Thr Thr Ile Gly Glu Ala Tyr Ile Pro 165 170 175 Ile AlaPhe Ala Ala Ala Ala Ala Ala Asp Pro Asp Ala Lys Leu Phe 180 185 190 TyrAsn Asp Tyr Asn Leu Glu Tyr Gly Gly Ala Lys Ala Ala Ser Ala 195 200 205Arg Ala Ile Val Gln Leu Val Lys Asn Ala Gly Ala Lys Ile Asp Gly 210 215220 Val Gly Leu Gln Ala His Phe Ser Val Gly Thr Val Pro Ser Thr Ser 225230 235 240 Ser Leu Val Ser Val Leu Gln Ser Phe Thr Ala Leu Gly Val GluVal 245 250 255 Ala Tyr Thr Glu Ala Asp Val Arg Ile Leu Leu Pro Thr ThrAla Thr 260 265 270 Thr Leu Ala Gln Gln Ser Ser Asp Phe Gln Ala Leu ValGln Ser Cys 275 280 285 Val Gln Thr Thr Gly Cys Val Gly Phe Thr Ile TrpAsp Trp Thr Asp 290 295 300 Lys Tyr Ser Trp Val Pro Ser Thr Phe Ser GlyTyr Gly Ala Ala Leu 305 310 315 320 Pro Trp Asp Glu Asn Leu Val Lys LysPro Ala Tyr Asn Gly Leu Leu 325 330 335 Ala Gly Met Gly Val Thr Val ThrThr Thr Thr Thr Thr Thr Thr Ala 340 345 350 Thr Ala Thr Gly Lys Thr ThrThr Thr Thr Thr Gly Ala Thr Ser Thr 355 360 365 Gly Thr Thr Ala Ala HisTrp Gly Gln Cys Gly Gly Leu Asn Trp Ser 370 375 380 Gly Pro Thr Ala CysAla Thr Gly Tyr Thr Cys Thr Tyr Val Asn Asp 385 390 395 400 Tyr Tyr SerGln Cys Leu Ser Ile Ala Gln Pro Lys Pro Ala Gly Val 405 410 415 Leu AlaIle Gln Ser Val Arg Phe Ile Tyr His Asn Thr Gln Asn Ser 420 425 430 LeuLeu Asp Leu Lys Lys Lys 435 927 base pairs nucleic acid single linearDNA (genomic) unknown CDS join(31..723, 727..849, 853..900, 904..927) 5TCCCTTCTAC TTAGTATTCA CTGACTTACC ATG GCT CGC CTA TCT CAG TTC CTT 54 MetAla Arg Leu Ser Gln Phe Leu 1 5 CTG GCC TGC GCT CTT GCA GTC AAA GCC TTCGCT GCC CCC GCC GCC GAG 102 Leu Ala Cys Ala Leu Ala Val Lys Ala Phe AlaAla Pro Ala Ala Glu 10 15 20 CCC GTC GAG GAA CGG GGC CCT AAC TTC TTT TCTGCC CTT GCT GGG CGC 150 Pro Val Glu Glu Arg Gly Pro Asn Phe Phe Ser AlaLeu Ala Gly Arg 25 30 35 40 TCG ACT GGC AGC TCC ACT GGC TAC TCG AAC GGCTAT TAC TAT AGC TTC 198 Ser Thr Gly Ser Ser Thr Gly Tyr Ser Asn Gly TyrTyr Tyr Ser Phe 45 50 55 TGG ACC GAT GGC GCA AGC GGC GAT GTT GAA TAC AGCAAC GGC GCC GGG 246 Trp Thr Asp Gly Ala Ser Gly Asp Val Glu Tyr Ser AsnGly Ala Gly 60 65 70 GGG TCC TAC AGC GTG ACC TGG TCA TCG GCC TCG AAC TTCGTC GGT GGA 294 Gly Ser Tyr Ser Val Thr Trp Ser Ser Ala Ser Asn Phe ValGly Gly 75 80 85 AAG GGC TGG AAC CCT GGA AGT GCT CAT GAC ATT ACG TAC TCCGGC TCC 342 Lys Gly Trp Asn Pro Gly Ser Ala His Asp Ile Thr Tyr Ser GlySer 90 95 100 TGG ACC AGC ACA GGA AAT AGC AAC AGC TAC CTC TCC GTC TACGGC TGG 390 Trp Thr Ser Thr Gly Asn Ser Asn Ser Tyr Leu Ser Val Tyr GlyTrp 105 110 115 120 ACC ACC GGT CCT CTC GTC GAG TAC TAT ATC CTG GAG GACTAC GGG GAG 438 Thr Thr Gly Pro Leu Val Glu Tyr Tyr Ile Leu Glu Asp TyrGly Glu 125 130 135 TAC AAC CCC GGC TCA GCT GGC ACT TAC AAA GGC TCG GTCTAC TCC GAC 486 Tyr Asn Pro Gly Ser Ala Gly Thr Tyr Lys Gly Ser Val TyrSer Asp 140 145 150 GGA TCG ACA TAC AAT ATC TAC ACG GCG ACC CGC ACC AACGCC CCC TCC 534 Gly Ser Thr Tyr Asn Ile Tyr Thr Ala Thr Arg Thr Asn AlaPro Ser 155 160 165 ATC CAG GGC ACG GCC ACT TTC ACG CAG TAC TGG TCC ATTCGC CAG ACA 582 Ile Gln Gly Thr Ala Thr Phe Thr Gln Tyr Trp Ser Ile ArgGln Thr 170 175 180 AAG CGC GTC GGC GGT ACC GTG ACG ACT GCC AAC CAT TTCAAT GCC TGG 630 Lys Arg Val Gly Gly Thr Val Thr Thr Ala Asn His Phe AsnAla Trp 185 190 195 200 GCG AAG CTG GGA ATG AAT CTG GGC ACG CAC AAC TATCAG ATC GTC GCT 678 Ala Lys Leu Gly Met Asn Leu Gly Thr His Asn Tyr GlnIle Val Ala 205 210 215 ACT GAA GGC TAC TAC TCG TCT GGG TCT GCG TCC ATTACG GTT GCC 723 Thr Glu Gly Tyr Tyr Ser Ser Gly Ser Ala Ser Ile Thr ValAla 220 225 230 TGA GAG CGT GCA GAT ATC CTG CTG CGA TAT ATG CTG TAT CTCTGG CAC 771 Glu Arg Ala Asp Ile Leu Leu Arg Tyr Met Leu Tyr Leu Trp His235 240 245 CGT TTC TGT GAT GGC AAT GAG TGG ATG AGG AAG TTG GCT TGT TCGTAC 819 Arg Phe Cys Asp Gly Asn Glu Trp Met Arg Lys Leu Ala Cys Ser Tyr250 255 260 ATG AGC AGG GTG GTA GTA TCG GAA TTT GGA TGA GCA TTG GAT TTCGAA 867 Met Ser Arg Val Val Val Ser Glu Phe Gly Ala Leu Asp Phe Glu 265270 275 TTA TTT TTT ATT CAA TCT CAG CCT CCA GTT TCG TAG CAA CAA GTA AAA915 Leu Phe Phe Ile Gln Ser Gln Pro Pro Val Ser Gln Gln Val Lys 280 285290 AAA AAA AAA AAA 927 Lys Lys Lys Lys 295 296 amino acids amino acidlinear protein unknown 6 Met Ala Arg Leu Ser Gln Phe Leu Leu Ala Cys AlaLeu Ala Val Lys 1 5 10 15 Ala Phe Ala Ala Pro Ala Ala Glu Pro Val GluGlu Arg Gly Pro Asn 20 25 30 Phe Phe Ser Ala Leu Ala Gly Arg Ser Thr GlySer Ser Thr Gly Tyr 35 40 45 Ser Asn Gly Tyr Tyr Tyr Ser Phe Trp Thr AspGly Ala Ser Gly Asp 50 55 60 Val Glu Tyr Ser Asn Gly Ala Gly Gly Ser TyrSer Val Thr Trp Ser 65 70 75 80 Ser Ala Ser Asn Phe Val Gly Gly Lys GlyTrp Asn Pro Gly Ser Ala 85 90 95 His Asp Ile Thr Tyr Ser Gly Ser Trp ThrSer Thr Gly Asn Ser Asn 100 105 110 Ser Tyr Leu Ser Val Tyr Gly Trp ThrThr Gly Pro Leu Val Glu Tyr 115 120 125 Tyr Ile Leu Glu Asp Tyr Gly GluTyr Asn Pro Gly Ser Ala Gly Thr 130 135 140 Tyr Lys Gly Ser Val Tyr SerAsp Gly Ser Thr Tyr Asn Ile Tyr Thr 145 150 155 160 Ala Thr Arg Thr AsnAla Pro Ser Ile Gln Gly Thr Ala Thr Phe Thr 165 170 175 Gln Tyr Trp SerIle Arg Gln Thr Lys Arg Val Gly Gly Thr Val Thr 180 185 190 Thr Ala AsnHis Phe Asn Ala Trp Ala Lys Leu Gly Met Asn Leu Gly 195 200 205 Thr HisAsn Tyr Gln Ile Val Ala Thr Glu Gly Tyr Tyr Ser Ser Gly 210 215 220 SerAla Ser Ile Thr Val Ala Glu Arg Ala Asp Ile Leu Leu Arg Tyr 225 230 235240 Met Leu Tyr Leu Trp His Arg Phe Cys Asp Gly Asn Glu Trp Met Arg 245250 255 Lys Leu Ala Cys Ser Tyr Met Ser Arg Val Val Val Ser Glu Phe Gly260 265 270 Ala Leu Asp Phe Glu Leu Phe Phe Ile Gln Ser Gln Pro Pro ValSer 275 280 285 Gln Gln Val Lys Lys Lys Lys Lys 290 295 20 base pairsnucleic acid single linear cDNA unknown 7 CATCAACATT CATTCATTCA 20 20base pairs nucleic acid single linear cDNA unknown 8 TTTAATTCATTCCTCAAGCT 20 20 base pairs nucleic acid single linear cDNA unknown 9CAAGAGCAGT CATCCCTTCT 20 20 base pairs nucleic acid single linear cDNAunknown 10 TTCCAACATG GTTCAAATCA 20 20 base pairs nucleic acid singlelinear cDNA unknown 11 AAGCAGCTGC TCTGGCTGTC 20 20 base pairs nucleicacid single linear cDNA unknown 12 CTTTTCGCCA GCAATGTGCT 20 21 basepairs nucleic acid single linear cDNA unknown 13 CTCCAACCCC ATCGAGCCCC G21 21 base pairs nucleic acid single linear cDNA unknown 14 CCAGGCCTCGGTGAGCATCG A 21 21 base pairs nucleic acid single linear cDNA unknown 15TGCCAAATTA CAAGGCGCAC G 21 21 base pairs nucleic acid single linear cDNAunknown 16 CAAGAAGTAC CTGGGCACCA T 21 20 base pairs nucleic acid singlelinear cDNA unknown 17 GAACCCCCAC AATCACGCAA 20 20 base pairs nucleicacid single linear cDNA unknown 18 AAATGGTCGG ACTGCTTTCA 20 19 basepairs nucleic acid single linear cDNA unknown 19 ATCACCGCGG CGCTTGCCG 1920 base pairs nucleic acid single linear cDNA unknown 20 CTGTGTTGCCAAACATTGTC 20 20 base pairs nucleic acid single linear cDNA unknown 21TCTGCCGTTG GTCTGGATCA 20 20 base pairs nucleic acid single linear cDNAunknown 22 GGCTGCAGTT GCCAAAGGAC 20 20 base pairs nucleic acid singlelinear cDNA unknown 23 TTCAATACTT TGGCACAGCT 20 20 base pairs nucleicacid single linear cDNA unknown 24 ACGGATAATC CCGAGCTCAC 20 21 basepairs nucleic acid single linear cDNA unknown 25 GGATATTCCA TACGTTACTC A21 20 base pairs nucleic acid single linear cDNA unknown 26 GCTGAACAACACCGCGGACT 20 23 base pairs nucleic acid single linear cDNA unknown 27TTGGTCAAAT TACCCCTGGA AAC 23 20 base pairs nucleic acid single linearcDNA unknown 28 TCGATGAAGT GGGATGCCAC 20 22 base pairs nucleic acidsingle linear cDNA unknown 29 AGAACCATCT CAGGGCACCT TC 22 15 base pairsnucleic acid single linear cDNA unknown 30 ACGTTCACGA AAGGC 15 17 basepairs nucleic acid single linear cDNA unknown 31 CTTCTACTTA GTATTCA 1720 base pairs nucleic acid single linear cDNA unknown 32 CTGACTTACCATGGCTCGCC 20 20 base pairs nucleic acid single linear cDNA unknown 33TATCTCAGTT CCTTCTGGCC 20 19 base pairs nucleic acid single linear cDNAunknown 34 TGCGCTCTTG CAGTCAAAG 19 20 base pairs nucleic acid singlelinear cDNA unknown 35 CCTTCGCTGC CCCCGCCGCC 20 20 base pairs nucleicacid single linear cDNA unknown 36 GAGCCCGTCG AGGAACGGGG 20 20 basepairs nucleic acid single linear cDNA unknown 37 CCCTAACTTC TTTTCTGCCC20 19 base pairs nucleic acid single linear cDNA unknown 38 TTGCTGGGCGCTCGACTGG 19 21 base pairs nucleic acid single linear cDNA unknown 39CAGCTCCACT GGCTACTCGA A 21 204 base pairs nucleic acid single linearcDNA unknown 40 CATCAACATT CATTCATTCA TTTAATTCAT TCCTCAAGCT CAAGAGCAGTCATCCCTTCT 60 TTCCAACATG GTTCAAATCA AAGCAGCTGC TCTGGCTGTC CTTTTCGCCAGCAATGTGCT 120 CTCCAACCCC ATCGAGCCCC GCCAGGCCTC GGTGAGCATC GATGCCAAATTCAAGGCGCA 180 CGGCAAGAAG TACCTGGGCA CCAT 204 265 base pairs nucleicacid single linear cDNA unknown 41 AAAATGGTCG GACTGCTTTC AATCACCGCGGCGCTTGCCG CGACTGTGTT GCCAAACATT 60 GTCTCTGCCG TTGGTCTGGA TCAGGCTGCAGTTGCCAAAG GACTTCAATA CTTTGGCACA 120 GCTACGGATA ATCCCGAGCT CACGGATATTCCATACGTTA CTCAGCTGAA CAACACCGCG 180 GACTTTGGTC AAATTACCCC TGGAAACTCGATGAAGTGGG ATGCCACAGA ACCATCTCAG 240 GGCACCTTCA CGTTCACGAA AGGCG 265 179base pairs nucleic acid single linear cDNA unknown 42 TCCCTTCTACTTAGTATTCA CTGACTTACC ATGGCTCGCC TATCTCAGTT CCTTCTGGCC 60 TGCGCTCTTGCAGTCAAAGC CTTCGCTGCC CCCGCCGCCG AGCCCGTCGA GGAACGGGGC 120 CCTAACTTCTTTTCTGCCCT TGCTGGGCGC TCGACTGGCA GCTCCACTGG CTACTCGAA 179

What is claimed is:
 1. An enzyme preparation comprising (a) anessentially pure enzym having xylanase activity, as measured by releaseof reducing sugars from birch xylan or by release of blue color fromAZCL-Birch xylan, said enzyme having a pH optimum of 4-5 and atemperature optimum of 70-80° C., which is encoded by a DNA sequencewhich hybridizes to a DNA sequence depicted in SEQ ID NO. 3 under thefollowing conditions: hybridizing in 5×SSC, 5×Denhardt's solution, 50 mMsodium phosphate, pH 6.8 and 50 ug denatured sonicated calf thymus DNAfor 18 hrs. at about 40° C. followed by warming tbree times in 2×SSC,0.2% SDS at 40° C. for 30 minutes and (b) a second enzyme havingcellulolytic, xylanolytic or pectinolytic activity.
 2. The enzymepreparation according to claim 1, wherein said essentially pure enzymehas a molecluar weight of about 56 kDa.
 3. The enzyme preparationaccording to claim 1, wherein said essentially pure enzyme has anapparent pI in the range of about 4.5-4.7.
 4. The enzyme preparationaccording to claim 1, wherein said essentially pure enzyme is derivablefrom a filamentous fungus or yeast.
 5. The enzyme preparation accordingto claim 1, wherein said essentially pure enzyme is derivable from afungal strain of Aspergilus, Trichoderma, Penicillium, Fusarium orHumicola.
 6. The enzyme preparation according to claim 1, wherein saidessentially pure enzyme is derivable from a strain of Asperigillusarculeatus, Aspergillus niger or Asperigillus oryzae.
 7. The enzymepreparation according to claim 1 wherein said essentially pure enzyme isencoded by a DNA sequence isolated from a DNA library of Aspergillusaculeatus, CBS 101.43.
 8. The enzyme preparation according to claim 1,wherein said second ensyme is producible from Aspergillus, Humicola orTrichoderma.
 9. The enzyme preparation according to claim 1, whereinsaid second enyme is CELUZYME™.
 10. The enzyme preparation according toclaim 1, wherein said second enzyme is CELLUCLAST™.
 11. An enzymepreparation comprising an enzyme having xylanase activity, as measuredby release of reducing sugars from birch xylan or by release of bluecolor from AZCL-Birch xylan, said enzyme has a pH optimum of 4-5; (b) atemperature optimum of 70-80° C.; (c) is encoded by a DNA sequencecomprising thc partial DNA sequnce depicted in SEQ ID NO. 41; (d) isderivable from a strain of Aspergillus and (b) a second enzyme havingcellulytic, xylanolytic or pectinolytic activity.
 12. The preparationaccording to claim 11, wherein said essentially pure enzyme has amolecular weight of about 56 kDa.
 13. The enzyme preparation accordingto claim 11, wherein said essentially pure enzyme has an apparent pI inthe range of about 4.5-4.7.
 14. The enzyme preparation according toclaim 11, wherein said essentially pure enzyme is derivable from afilamentous fungus or yeast.
 15. The enzyme preparation according toclaim 11, wherein said essentially pure enzyme is derivable from afungal strain of Aspergillus, Trichoderma, Penicillum, Fusarium orHumicola.
 16. The enzyme preparation according to claim 11, wherein saidessentially pure enzym is derivable from a strain of Aspergillusaculeatus, Aspergillus niger or Aspergillus oryzae.
 17. The enzymepreparation according to claim 11, wherein said essentially pure enzymeis encoded by a DNA sequence isolated from a DNA library of Aspergillusaculeatus, CBS 101.43.
 18. The enzyme preparation according to claim 11,wherein said second enzyme is producible fom Aspergillus, Humicola orTrichoderma.
 19. The enzyme preparation according to claim 11, whereinsaid second enzyme is CELLUZYME™.
 20. The enzyme preparation accordingto claim 11, wherein said second enzyme is CELLUCLAST™.